Bispecific fab fusion proteins comprising a cd3-binding fab fragment with n-terminal fusion to a binding domain and methods of use

ABSTRACT

The present disclosure relates generally to multi-specific Fab fusion proteins (MSFP) which comprise an antibody Fab fragment with both N-termini fused to a fusion moiety (fusion moiety A or B). MSFP containing the Fab fragment exhibit significantly reduced binding ability of the Fab fragment to the Fab target. Binding of the Fab to its target is restored when the MSFP is clustered on a cell surface by binding of the fusion moieties to their target. The reduced binding of the Fab to its target, especially when presented on a cell surface in its native state, absent fusion moiety binding provides advantages such as: reduced side effects and allows desirable pharmacological effects of selectivity and specificity in a controlled manner.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 14/479,203, filed Sep. 5, 2014, now U.S. Pat. No.11,013,800, issued May 25, 2021, which is a continuation of U.S. patentapplication Ser. No. 13/473,017, filed May 16, 2012, now U.S. Pat. No.8,846,042, issued Sep. 30, 2014, which claims priority to U.S.Provisional Application No. 61/486,690, filed May 16, 2011, the contentsof each of which are incorporated herein by reference in theirentireties.

STATEMENT REGARDING SEQUENCE LISTING

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 720622000710SEQLIST.TXT,date recorded: Apr. 9, 2021, size: 188 KB).

BACKGROUND Technical Field

The present disclosure relates generally to multi-specific Fab fusionproteins (MSFP). In particular, the MSFP of the present disclosurecomprise an antibody Fab fragment with both N-termini fused to a fusionmoiety by a cleavable or non-cleavable linker. The Fab fragment of theMSFP specifically binds to native cell surface target antigens as wellas certain soluble forms of the same antigen. One or both fusionmoieties bind specifically to target antigens on cell surfaces.

Description of the Related Art

Conventional immunoglobulin G (IgG) is a tetrameric molecule comprisingtwo identical immunoglobulin heavy chains and two identicalimmunoglobulin light chains. IgG heavy chain has a variable region atthe N-terminus followed by the first constant region (CH1), a hinge andtwo additional constant regions (CH2CH3). IgG light chain is comprisedof two domains: an N-terminal variable region and a C-terminal constantregion. The heavy chain variable region (VH) interacts with the lightchain variable region (VL) to constitute the minimal antigen bindingregion, Fv. The antigen binding region is stabilized by the interactionbetween the first constant region of heavy chain (CH1) and the lightconstant (CL) and further by the formation of a disulfide bond betweenthe two constant regions (to form a Fab fragment). The homodimerizationof CH2CH3 domains (to form Fc) and consequently the hinge disulfide bondformation stabilize the IgG structure. Thus an IgG has two antigenbinding Fab arms which are relatively flexible in orientation with eachother and with the Fc domain. In addition, the binding regions (whichinteract directly with antigen) are located at the N terminus with nofurther structures beyond. Conceivably, this structure featurefacilitates antibody interaction with antigen with minimal interferencefrom steric hindrance. This property is especially important for bindingto cell surface antigen that is often located very close to the complexcell membrane.

In recent years, full length monoclonal antibodies have beensuccessfully used to treat cancer, autoimmune and inflammatory diseasesand other human diseases (Nelson, Nat. review, Drug Discovery (2010)9:767-74). Although five different types of immunoglobulins (IgA, IgD,IgG, IgM and IgE) exist naturally, IgG represents the most suitablemodality for human therapeutics because of the favorable propertiesincluding high binding affinity and specificity, high bioavailability,long serum half life in circulation, potential effector functioncapability and the industrial-scale manufacturability.

Monoclonal antibodies (non conjugated or naked antibody) currentlyapproved by drug regulatory agencies worldwide for clinical use inoncology setting working generally by one or a combination of thefollowing mechanisms: 1) blocking cell growth signaling, 2) blocking theblood supply to cancer cells, 3) directly mediating cell apoptosis, 4)eliciting immunological effector functions such as antibody dependentcellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis(ADCP) and complement dependent cytotoxicity (CDC), and 5) promotingadaptive immunity towards tumors.

Monoclonal antibody therapies have demonstrated survival benefits in theclinic. However, the overall response rates in cancer patients are low,and the survival benefits are marginal (several months) compared tochemotherapy. Although the underlying reasons for the lack of robustclinical anti-cancer activities are not fully understood, research hassuggested that cancer cells often quickly develop compensating signalingpathways to escape cell death. Also, cancer stem cells (CSC), which areconsidered as potent cancer initiating cells, are less active at cellproliferation therefore they tend to sustain the lack of growth signalbetter.

In recent years, ADCC was demonstrated to play a significant role in theclinical efficacy of anti-cancer antibodies. Antibody Fc binds to Fcgamma receptors of which there are numerous forms: FcγRI, FcγRIIa,FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. The Fc domain has especiallyhigh affinity for FcγRIIIa, which is expressed on natural killing (NK)cells, macrophages, and neutrophils. Binding of Fc to FcγRIIIa activatesNK cells which can then destroy nearby cancer cells.

Engineered antibodies with enhanced activating FcγR binding propertiesvia either protein engineering (involving a variety of amino acidmutations in the CH2 region) or production host cell (CHO) lineengineering to reduce or eliminate fucosylation in the Asn297 glycanstructure have been successfully tested in preclinical studies withimproved biological activities. IgG antibodies containing enhancedeffector functions are currently in clinical testing with the goal ofimproved efficacy. The current available clinical data indicate thatthese antibodies are very promising.

Another anti-cancer therapeutic approach is to utilize T cells. T cellsprovide defense against cancer throughout life by patrolling the body insearch for newly arisen cancer cells and eliminating them effectivelyand promptly. Successful therapeutic approaches harnessing T cellimmunity in cancer treatment include: 1) the FDA approved use ofProleukin (recombinant IL-2) for metastatic melanoma and metastatickidney cancer; 2) FDA approved use of PROVENGE® (Sipuleucel-T) forasymptomatic metastatic hormone refractory prostate cancer. PROVENGE® isa dendritic cell vaccine that activates prostate cancer-specificcytotoxic T cells ex vivo, which cells are then reinfused into thepatient; 3) FDA approve use of ipilimumab (anti CTLA-4 antibody toactivate T cells by inhibiting T cell inhibitory signaling pathway) foradvanced melanoma.

Because T cells do not express Fc gamma receptors (FcγR), anti-tumorantibodies cannot effectively activate cytotoxic T cells directly. Oneof the many promising methods aimed to activate T cell for tumor killingpurposes is to use bispecific antibodies (bsAb) to directly bring Tcells to the proximity of tumor cells, resulting in activation of Tcells and the killing the tumor cells. CD28 and CD137 (4-1BB) are twopotent T cell co-stimulatory receptors utilized in bispecific targetingapproaches. Examples include: CD28×NG2 (Grosse-Hovest et al., Eur JImmunol 33:1334-40, 2003), CD28×CD20 (Otz et al., Leukemia 23:71-7,2009) and 4-1BB×CD20 (Liu et al., J Immunother 33:500-9, 2010). Other Tcell surface targets capable of triggering T activation have also beenused for retargeting them to tumor cells using bispecific antibodies.Various bispecific and multispecific antibody formats have beendeveloped in the past and reviewed recently (Muller and Kontermann,Biodrugs 24: 89-98, 2010; Chames and Baty, mAbs 1:539-47, 2009; Deyevand Lebedenko, BioEssays 30:904-918, 2008). These formats fall into thefollowing three large categories: 1) IgG-like bispecific molecules basedon Fc heterodimerization or covalent fusion to the heavy or light chain,including quadroma technology (Staerz et al., PNAS 83:1453-7, 1986),knob and hole technology (Nat Biotechnol. 16: 677-81; J. Biol. Chem.285:19637-46, 2010), strand-exchange engineered domain “SEED” technology(Davis et al., PEDS 23:195-202, 2010), fusion to the C-terminus of IgGheavy or light chain (Coloma and Morrison, Nat. Biotechnol. 15:159-63,1997; Shen et al., J. Immunol. Methods 318:65-74, 2007; Orcutt et al.,PEDS 23:221-8, 2010; Dong et al., J. Biol. Chem., 286:4703-17, 2011,Lazar et al., patent application, US20110054151), fusion to theN-terminus an IgG heavy or light chain (Shen, et al., J. Biol. Chem.281:10706-17, 2006; Wu et al., Nat. Biotechnol. 25:1290-7, 2007), 2) Fcfusion bispecific antibodies (Mabry et al., PEDS 23:115-127, 2010;Miller et al., PEDS 23:549-557, 2010), 3) antibody variable region onlymolecules through fusion or noncovalent association, including diabody(Db) (Holliger et al., PNAS 90:6444-8, 1993), disulfide bond linkeddiabody (also known as dual affinity re-targeting or DART) (Johnson etal., J. Mol. Biol. 399:436-49, 2010), single chain diabody (scDb) (Alt,et al., FEBS Letters 454:90-4, 1999), tandem diabody (tandAbs)(Kipriyanov, et al., J. Mol. Biol. 293:41-56, 1999), tandem single chainFv (taFv) (Mack et al., PNAS 92:7021-5, 1995), and 4) Fab based fusionmolecules, including bibody and TR1BODY™ (Schoonjans et al., J. Immunol.165:7050-7, 2000; Website of Biotecnol SA, at the world-wide web addressbiotecnol.com), Fab fusion to single domain antibodies (patentapplication, US2010/0239582A1), and Fab′2-fusion (US patent, U.S. Pat.No. 5,959,083).

Efforts in the area of the bispecific antibody field over the last twodecades started to bear fruits clinically. Catumaxomab (REMOVAB®, ananti-CD3, anti-EpCAM trifunctional antibody), was approved in Europe forsymptomatic malignant ascites in 2009. However, while bispecificantibodies have demonstrated potent tumor cell killing potential, severeside effects, including systemic immune activation, immunogenicity(anti-drug antibody response) and general poor manufacturability ofthese molecules remain and to a large extent, have prevented this classof drugs from broad applications. Recently bispecific antibodytechnology platform referred to as bi-specific T cell engagers, orBITE®, employing an anti-CD19 scFv-anti-CD3 scFv fusion (Blinatumomab),attracted a lot of attention because of its outstanding potencydemonstrated in preclinical and clinical tests (Bargou et al., Science(2008) 321:974-7). In particular, patients with non-Hodgkin's lymphomashowed tumor regression, and in some cases complete remission during aclinical trial of blinatumomab administration. However, blinatumomabcaused severe side effects including central nervous system side effectsmanifested by the loss of language ability and disorientation. Symptomswere transient and reversible once administration of the drug wasstopped. It was hypothesized that the direct binding of the CD3 (on Tcells) by the drug causing T partial activation and cell redistribution(patent application, US2010/0150918A1). Some of the redistributed Tcells adhere to the brain micro-vasculature, partially activate theendothelial cells and lead to the enhanced permeability of themicro-vasculature in the brain. It was observed in the clinic trial thatthe incidence of side effects was lower in patients with high B cell toT cell ratios than those with low ratios. It has also been reported thatusing different CD3 binding antibody fragments can alleviate or avoid Tcell redistribution in Monkeys. These observations strongly suggest thatantibody binding to CD3 together with the binding epitope on CD3 and theresultant partial activation of the T cell may be the direct cause ofthe severe CNS side effects.

Another drawback of the CD19×CD3 bispecific scFv-scFv fusion is that thedrug requires daily intravenous infusion (i.v.) drug delivery due to itsshort half-life and incompatibility with subcutaneous administration. Inaddition, scFv-scFv fusion proteins have a tendency to aggregate.Therefore, BiTE molecules require highly complicated antibodyengineering skills and it is laborious to make them stable andmanufacturable.

Anti-cancer activities of antibody drugs engaging FcγR or CD3-expressingimmune cells demonstrated clinical proof of concept. However, theshortcomings of the current bispecific antibody formats remain achallenge for broad application of these drugs for treating cancerpatients with good efficacy and safety profiles. Therefore, thereremains an urgent need for new bispecific molecule designs with improvedprofiles on product efficacy, stability, safety and manufacturability.

References for further background information include: Coloma andMorrison, Nat. Biotechnol. 15:159-63, 1997; Kontermann, Acta Pharmacol.Sin., 26, 1-9. 2005; Marvin and Zhu; Acta Pharmacol. Sin., 26,649-658.2005; Shen et al., J. Immunol. Methods, 318:65-74, 2007; Shen etal. JBC 281:10706-10714, 2006; Wu et al., Nat. Biotechnol., 25,1290-1297, 2007; Orcutt Prot PEDS 23:221-8, 2010; Mabry PEDS vol. 23 no.3 pp. 115-127, 2010; Schoonjans, The Journal of Immunology, 2000, 165:7050-7057; Michaelson, mAbs 1:2, 128-141; 2009; Robinson et al., BritishJournal of Cancer (2008) 99, 1415-1425.

BRIEF SUMMARY

One aspect of the present disclosure provides a multi-specific Fabfusion protein comprising: a Fab fragment that binds to a targetantigen; a first fusion moiety coupled at the N-terminus of the VL ofthe Fab fragment; and/or a second fusion moiety coupled at theN-terminus of the VH of the Fab fragment. In one embodiment of themulti-specific Fab fusion protein, the binding of the Fab fragment tothe Fab target antigen is reduced by between 50% and 90% as compared tothe binding of an identical Fab fragment to the Fab target in theabsence of the first and second fusion moieties.

One aspect of the present disclosure provides a multi-specific Fabfusion protein comprising a) a Fab fragment that binds to a targetantigen, wherein the Fab fragment comprises, i) an immunoglobulin lightchain variable region (VL) and an immunoglobulin light chain constantregion (CL); and ii) an immunoglobulin heavy chain variable region (VH),and an immunoglobulin heavy chain constant region 1 (CH1); wherein theCL and CH1 regions are optionally connected by a disulfide bond; b) afusion moiety A wherein the C-terminus of fusion moiety A is covalentlylinked to the N-terminal end of the VH domain; or c) a fusion moiety B,wherein the C-terminus of fusion moiety B is covalently linked to theN-terminal end of the VL domain; or both b and c; d) optionally, a firstlinker situated between the fusion moiety A and the VH domain; and e)optionally, a second linker situated between the fusion moiety B and theVL domain. In one embodiment of the MSFP, the fusion moiety A comprisesa binding domain and/or the fusion moiety B comprises a binding domain.The fusion moiety A and the fusion moiety B may be identical in sequenceor may comprise different sequences.

In yet a further embodiment of the multi-specific Fab fusion protein ofthe present disclosure, the fusion moiety A comprises a first bindingdomain and the fusion moiety B comprises a second binding domain,wherein the first and second binding domains bind to the same cellsurface antigen. In one embodiment, the fusion moiety A comprises afirst binding domain and the fusion moiety B comprises a second bindingdomain, wherein the first and second binding domains bind to differentcell surface antigens. In certain embodiments, the fusion moiety Acomprises a first binding domain and the fusion moiety B comprises asecond binding domain, wherein the first and second binding domains bindto the same epitope or to different epitopes.

In one embodiment of the multi-specific Fab fusion proteins of presentdisclosure, the Fab fragment binds to a cell surface target antigen. Inone particular embodiment, the fusion moiety A comprises a bindingdomain and the Fab fragment binds to the same cell surface antigen asthe binding domain. In another embodiment, the fusion moiety B comprisesa binding domain and the Fab fragment binds to the same cell surfaceantigen as the binding domain.

In certain embodiments of the multi-specific Fab fusion proteindescribed herein, binding of the Fab fragment to the antigen isinhibited by steric hindrance caused by the fusion moiety A and thefusion moiety B. In some embodiments, the steric hindrance results in nodetectable binding of the Fab fragment to the antigen. In otherembodiments, the binding of the Fab fragment to its target is reduced bybetween 50% and 90% as compared to the binding of an identical Fabfragment to its target in the absence of the fusion moiety A and thefusion moiety B.

In one embodiment of the multi-specific Fab fusion proteins describedherein, the first and second linkers are cleavable. In furtherembodiments, binding of the Fab fragment to the antigen is detectable ifthe first linker is cleaved. In another embodiment, binding of the Fabfragment to the antigen is detectable if the second linker is cleaved.In yet further embodiments, binding of the Fab fragment to the antigenis detectable if the first linker and the second linker are bothcleaved.

In another embodiment of the multi-specific Fab fusion proteinsdescribed herein, the immunoglobulin heavy chain constant region type isselected from α, δ, ε, γ, and μ. In one embodiment, the immunoglobulinheavy chain is derived from an IgG antibody and in further embodiments,the isotype of the IgG antibody is selected from IgG1, IgG2, IgG3 andIgG4.

In another embodiment of the multi-specific Fab fusion proteinsdescribed herein, the immunoglobulin light chain VL and CL are selectedfrom immunoglobulin kappa light chain and the immunoglobulin lambdalight chain.

In one embodiment, the fusion moiety A comprises a first binding domainwhich comprises an scFv antigen-binding domain. In one embodiment of themulti-specific Fab fusion proteins described herein, the fusion proteindoes not comprise the first linker or the second linker. In this regard,in some embodiments, the fusion moiety A has from 1-3 C-terminal aminoacid residues deleted; or wherein the fusion moiety B has from 1-3C-terminal amino acid residues deleted; or wherein both the fusionmoiety A and the fusion moiety B have from 1-3 C-terminal amino acidresidues deleted. In further embodiments of the MSFP described hereinwherein there is no first or second linker, the N-terminal end of theFab VH may have from 1-3 amino acid residues deleted; or the N-terminalend of the Fab VL may have from 1-3 amino acid residues deleted; or boththe N-terminal end of the VH and the N-terminal end of the VL may havefrom 1-3 amino acid residues deleted. In further embodiments of the MSFPdescribed herein wherein there is no first or second linker, the fusionmoiety A and the fusion moiety B may have from 1-3 C-terminal amino acidresidues deleted; and both the N-terminal end of the Fab VH and theN-terminal end of the Fab VL may have from 1-3 amino acid residuesdeleted. In yet further embodiments, the MSFP described herein containsany combination of a linker and a deletion.

In one embodiment of the multi-specific Fab fusion protein describedherein, or a homodimer thereof, the first linker comprises a proteasenon-cleavable sequence and/or the second linker comprises a proteasecleavable sequence. In another embodiment of the multi-specific Fabfusion protein described herein, or a homodimer comprising same, thefirst linker comprises a protease cleavable sequence and/or the secondlinker comprises a non-cleavable sequence. In certain embodiments, thefirst and second linkers comprise protease cleavable sequences cleavableby the same protease. In other embodiments, the first and second linkerscomprise protease cleavable sequences cleavable by different proteases.In related embodiments, the protease cleavable sequence comprises aserine protease, a cysteine protease, a aspartate protease, or a matrixmetalloprotease (MMP) cleavable sequence. In certain embodiments, theprotease cleavable sequence is a MMP cleavable sequence. In this regard,said matrix metalloprotease cleavable sequence may be a matrixmetalloprotease 1 (MMP-1), a matrix metalloprotease 2 (MMP-2), a matrixmetalloprotease 9 (MMP-9), or a matrix metalloprotease 14 (MMP-14)cleavable sequence.

In another embodiment of the multi-specific Fab fusion proteinsdescribed herein, the first linker is 1 to 10 amino acids long or is 1to 20 amino acids long. In some embodiments, the second linker comprisesa protease non-cleavable sequence or comprises a protease cleavablesequence. In this regard, the protease cleavable sequence may be aserine protease, a cysteine protease, a aspartate protease, or a matrixmetalloprotease (MMP) cleavable sequence. In certain embodiments, theprotease cleavable sequence is a MMP cleavable sequence and in thisregard, may be a MMP cleavable sequence selected from a matrixmetalloprotease 1 (MMP-1), a matrix metalloprotease 2 (MMP-2), a matrixmetalloprotease 9 (MMP-9), or a matrix metalloprotease 14 (MMP-14)cleavable sequence. In certain embodiments, the second linker is 1 to 10amino acids long or is 1 to 20 amino acids long.

In another aspect of the present disclosure, the multi-specific Fabfusion protein comprising a) a Fab fragment that binds to a targetantigen, wherein the Fab fragment comprises, i) an immunoglobulin lightchain variable region (VL) and an immunoglobulin light chain constantregion (CL); and ii) an immunoglobulin heavy chain variable region (VH),and an immunoglobulin heavy chain constant region 1 (CH1); wherein theCL and CH1 regions are optionally connected by a disulfide bond; b) afusion moiety A wherein the C-terminus of fusion moiety A is covalentlylinked to the N-terminal end of the VH domain; c) a fusion moiety B,wherein the C-terminus of fusion moiety B is covalently linked to theN-terminal end of the VL domain; d) optionally, a first linker situatedbetween the fusion moiety A and the VH domain; and e) optionally, asecond linker situated between the fusion moiety B and the VL domain;wherein the fusion moiety A comprises a binding domain and/or the fusionmoiety B comprises a binding domain and further, either or both of thebinding domain binds to a cell surface antigen. In certain embodiments,the cell surface antigen is a tumor antigen. In certain embodiments, oneof the binding domains (e.g., of either fusion moiety A or fusion moietyB) binds to human serum albumin.

In another embodiment of the multi-specific Fab fusion proteinsdescribed herein, the Fab binds to a target antigen selected from agroup of FcγR I, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIa, NKG2D, CD3, CD25,CTLA-4, FAS, FGFR1, FGFR2, FGFR3, FGFR4, GITR, LTβR, TLR, TRAIL receptor1, TRAIL receptor 2, EGFR, Her2/neu, ErbB3, CD25, and CD28. Inparticular embodiments, the Fab binds to CD3. In another embodiment, theFab binds to T cell receptor. The Fab may be humanized. In certainembodiments, the humanized Fab is derived from OKT3, UCHT-1, or SP34. Inone embodiment, the Fab is derived from a fully human antibody which insome embodiments is generated from phage display, yeast display, orhuman antibody gene transgenic mice.

In one embodiment of the multi-specific Fab fusion proteins describedherein, the fusion moiety A comprises a first binding domain and thefusion moiety B comprises a second binding domain, wherein the first andsecond binding domains bind to the same cell surface antigen, andwherein the first and second binding domains bind to a target selectedfrom a group of: FcγRIIb, CD28, CTLA-4, FAS, FGFR1, FGFR2, FGFR3, FGFR4,GITR, LTβR, TLR, TRAIL receptor 1, TRAIL receptor 2, CEA, PSMA, BCMA,CAIX, cMet, EGFR1, Her2/neu, ErbB3, EpCAM, Folate receptor, Ephrinreceptor, CD19, CD20, CD30, CD33, CD40, CD37, CD38, and CD138.

In a further embodiment of the multi-specific Fab fusion proteinsdescribed herein, the fusion moiety A comprises a first binding domainand the fusion moiety B comprises a second binding domain, wherein thefirst and second binding domains bind to different cell surface antigensand wherein the first and second binding domains bind to a differenttarget selected from a group of: FcγRIIb, CD28, CTLA-4, FAS, FGFR1,FGFR2, FGFR3, FGFR4, GITR, LTβR, TLR, TRAIL receptor 1, TRAIL receptor2, CEA, PSMA, BCMA, CAIX, cMet, EGFR1, Her2/neu, ErbB3, EpCAM, Folatereceptor, Ephrin receptor, CD19, CD20, CD30, CD33, CD40, CD37, CD38, andCD138.

In certain embodiments, of the multi-specific Fab fusion proteins thefirst and/or the second binding domain is an antigen-binding fragment ofan antibody. In this regard, the antigen-binding fragment may beselected from the group consisting of an scFv, a CDR, a Fv, animmunoglobulin VL domain, an immunoglobulin VH domain, an immunoglobulinVL and a VH domain, a Fab, a camelid VHH, a dAb (domain antibody). Incertain embodiments, the antigen-binding fragment is humanized and inother embodiments, the antigen-binding fragment is derived from a mouse,rat, or rabbit monoclonal antibody. In some embodiments, theantigen-binding fragment is derived from a fully human antibody whichmay be generated from phage display, yeast display, or a human antibodygene transgenic mouse.

In further embodiments, the first and/or the second binding domain of anMSFP described herein is selected from the group consisting of aFibronectin 3 domain (Fn3), an ankyrin repeat, and an Adnectin.

In one embodiment of the multi-specific Fab fusion proteins describedherein, the first and/or second linker comprises the amino acid sequenceset forth in SEQ ID NO: 133. (PLGLAG).

One aspect of the present disclosure provides isolated polynucleotidesencoding the multi-specific Fab fusion proteins described herein, andexpression vectors comprising the isolated polynucleotides, and isolatedhost cells comprising such vectors.

Another aspect of the present disclosure provides a method of expressinga multi-specific Fab fusion protein by culturing a host cell comprisingan isolated polynucleotide encoding an a multi-specific Fab fusionprotein as described herein under conditions in which the isolatedpolynucleotide expresses the encoded multi-specific Fab fusion protein.

A further aspect of the disclosure provides a pharmaceutical compositioncomprising one or more multi-specific Fab fusion proteins as describedherein, and a pharmaceutically acceptable carrier.

Another aspect of the disclosure provides a method of treating acondition comprising administering an effective amount of apharmaceutical composition comprising one or more multi-specific Fabfusion proteins as described herein, and a pharmaceutically acceptablecarrier, to a subject in need, wherein the condition is associated withan antigen to which the multi-specific Fab fusion protein can bind.

In a further embodiment of the multi-specific Fab fusion proteinsdescribed herein, the fusion moiety A and the fusion moiety B do notdimerize.

Another aspect of the present disclosure provides a multi-specific Fabfusion protein comprising: a Fab fragment that binds to the N-terminusof CD3 epsilon; a fusion moiety A linked to the N-terminus of a VL ofthe Fab fragment; or a fusion moiety B linked to the N-terminus of a VHof the Fab fragment; or both a fusion moiety A linked to the N-terminusof the VL of the Fab fragment and a fusion moiety B linked to theN-terminus of the VH of the Fab fragment. In one embodiment, the Fabfragment binds to an epitope within amino acids 1-27 of CD3 epsilon. Ina further embodiment, the Fab fragment cross-reacts with nonhumanprimate CD3 epsilon.

In one embodiment of the multi-specific Fab fusion proteins disclosedherein a. the Fab fragment comprises, i. an immunoglobulin light chainvariable region (VL) comprising the CDR1, CDR2 and CDR3 amino acidsequences set forth in SEQ ID NOs: 26-28 and an immunoglobulin lightchain constant region (CL); and ii. an immunoglobulin heavy chainvariable region (VH), comprising the CDR1, CDR2 and CDR3 amino acidsequences set forth in SEQ ID NOs: 23-25, and an immunoglobulin heavychain constant region 1 (CH1); wherein the CL and CH1 regions areoptionally connected by a disulfide bond; b. the C-terminus of thefusion moiety A is covalently linked to the N-terminal end of the VHdomain; or c. the C-terminus of the fusion moiety B is covalently linkedto the N-terminal end of the VL domain; or d. both b and c; e.optionally, a first linker situated between the fusion moiety A and theVH domain; and f. optionally, a second linker situated between thefusion moiety B and the VL domain. In one embodiment, the VH is selectedfrom any one of the amino acid sequences set forth in SEQ ID NOs: 34,38, 42, 46, 50, and 54. In another embodiment, VL is selected from anyone of the amino acid sequences set forth in SEQ ID NOs: 56, 58, 62, 66,and 70. In another embodiment, the fusion moiety A comprises a bindingdomain and/or the fusion moiety B comprises a binding domain. In oneembodiment, the fusion moiety A and the fusion moiety B are identical insequence. In yet another embodiment, the fusion moiety A and the fusionmoiety B comprise different sequences. In one particular embodiment, thefusion moiety A comprises a first binding domain and the fusion moiety Bcomprises a second binding domain, wherein the first and second bindingdomains bind to the same cell surface antigen. In certain embodiments,the fusion moiety A comprises a first binding domain and the fusionmoiety B comprises a second binding domain, wherein the first and secondbinding domains bind to different cell surface antigens. In otherembodiments, the fusion moiety A comprises a first binding domain andthe fusion moiety B comprises a second binding domain, wherein the firstand second binding domains bind to the same epitope. In anotherembodiment, the fusion moiety A comprises a first binding domain and thefusion moiety B comprises a second binding domain, wherein the first andsecond binding domains bind to different epitopes.

In one embodiment of the multi-specific Fab fusion proteins disclosedherein, binding of the Fab fragment to CD3 is inhibited by sterichindrance caused by the fusion moiety A and the fusion moiety B. In thisregard, the steric hindrance results in no detectable binding of the Fabfragment to CD3 in the absence of binding of the first and secondbinding domains to the cell surface antigen. In another embodiment, thebinding of the Fab fragment to CD3 is reduced by between 50% and 90% inthe absence of binding of the first and second binding domains to thecell surface antigen as compared to the binding of the Fab fragment toCD3 in the presence of binding of the first and second binding domainsto the cell surface antigen. In a further embodiment, the binding of theFab fragment to CD3 is reduced by between 50% and 90% as compared tobinding of an identical Fab fragment not having the fusion moiety A andthe fusion moiety B.

In another embodiment of the multi-specific Fab fusion proteinsdisclosed herein, the immunoglobulin heavy chain constant region type isselected from α, δ, ε, γ, and μ. In one embodiment, the immunoglobulinheavy chain is derived from an IgG antibody. In further embodiments, theisotype of the IgG antibody is selected from IgG1, IgG2, IgG3 and IgG4.In another embodiment, the immunoglobulin light chain VL and CL areselected from immunoglobulin kappa light chain and the immunoglobulinlambda light chain.

In one embodiment of the multi-specific Fab fusion proteins disclosedherein, the fusion moiety A comprises a first binding domain whichcomprises an scFv antigen-binding domain.

In another embodiment of the multi-specific Fab fusion proteins thefirst linker and the second linker are 1 to 20 amino acids long. In oneparticular embodiment, the first and/or second linkers compriseGly-Arg-Ala.

In one embodiment of the multi-specific Fab fusion proteins disclosedherein the Fab is humanized.

In yet another embodiment of the multi-specific Fab fusion proteinsdisclosed herein, the first and second binding domains bind to a targetselected from a group of: FcγRIIb, CTLA-4, FAS, FGFR1, FGFR2, FGFR3,FGFR4, GITR, LTβR, TLR, TRAIL receptor 1, TRAIL receptor 2, CD28, CEA,PSMA, BCMA, CAIX, cMet, EGFR1, Her2/neu, ErbB3, EpCAM, Folate receptor,Ephrin receptor, CD19, CD20, CD30, CD33, CD40, CD37, CD38, and CD138. Inone embodiment, the 1st and 2nd binding domains are selected from anyone of the amino acid sequences set forth in SEQ ID NOs: 78, 88, and 94.

In another embodiment, the first and second binding domains bind to adifferent target selected from the group consisting of: FcγRIIb, CTLA-4,FAS, FGFR1, FGFR2, FGFR3, FGFR4, GITR, LTβR, TLR, TRAIL receptor 1,TRAIL receptor 2, CD28, CEA, PSMA, BCMA, CAIX, cMet, EGFR1, Her2/neu,ErbB3, EpCAM, Folate receptor, Ephrin receptor, CD19, CD20, CD30, CD33,CD40, CD37, CD38, and CD138.

In one embodiment, the first and/or the second binding domain is anantigen-binding fragment of an antibody. In another embodiment, theantigen-binding fragment is selected from the group consisting of anscFv, a CDR, a Fv, an immunoglobulin VL domain, an immunoglobulin VHdomain, an immunoglobulin VL and a VH domain, a Fab, a camelid VHH, adAb. In yet a further embodiment, the antigen-binding fragment ishumanized. In other embodiments, the antigen-binding fragment is derivedfrom a mouse, rat, or rabbit monoclonal antibody, and theantigen-binding fragment may also be derived from a fully humanantibody. In this regard, the fully human antibody is generated fromphage display, yeast display, or a human antibody gene transgenic mouse.

In one embodiment, the antigen binding fragment: 1) comprises the VHCDRsand the VLCDRs set forth in SEQ ID NOs: 139-144, respectively; 2)comprises the VHCDRs and the VLCDRs set forth in SEQ ID NOs: 145-150,respectively; 3) comprises the VH present within the scFv as set forthin the amino acid sequence of any one of SEQ ID Nos: 78, 88, and 94; 4)comprises the VL present within the scFv as set forth in the amino acidsequence of any one of SEQ ID Nos: 78, 88, and 94; or 5) comprises anscFv selected from any one of the amino acid sequences set forth in SEQID NOs: 78, 88, and 94.

In certain embodiments, of the multi-specific Fab fusion proteins, theCL region comprises a knob or hole mutation and the CH1 region comprisesa corresponding knob or hole mutation such that the CL region and CH1region stably interact.

In another embodiment, the multi-specific Fab fusion proteins disclosedherein, comprise any one of the amino acid sequences selected from SEQID NOs: 84, 90, 96, and 100; and any one of the amino acid sequencesselected from SEQ ID NOs: 32, 60, 64, 68, and 72. In a furtherembodiment, the multi-specific Fab fusion protein of this disclosurecomprise any one of the amino acid sequences selected from SEQ ID NOs:86, 92, 98, and 102; and any one of the amino acid sequences selectedfrom SEQ ID NOs: 30, 36, 40, 44, 48, and 52. In certain embodiments, theCL and CH1 regions are connected by a disulfide bond.

Another aspect of the present disclosure provides an isolatedpolynucleotide encoding any of the multi-specific Fab fusion proteinsdisclosed herein, and expression vectors comprising the isolatedpolynucleotides. This disclosure also provides isolated host cellscomprising such vectors. The present disclosure also provides methods ofexpressing a multi-specific Fab fusion protein by culturing the hostcells under conditions in which the isolated polynucleotide expressesthe encoded multi-specific Fab fusion protein.

The present disclosure also provides pharmaceutical compositionscomprising the multi-specific Fab fusion proteins described herein and apharmaceutically acceptable carrier.

Another aspect of the present disclosure provides methods for treating acancer comprising administering an effective amount of a pharmaceuticalcomposition comprising the multi-specific Fab fusion proteins describedherein and a pharmaceutically acceptable carrier to a subject having thecancer or suspected of having the cancer, wherein the cancer isassociated with an antigen to which the multi-specific Fab fusionprotein can bind.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram depicting a multi-specific Fab fusion protein(MSFP). Fusion moiety A and fusion moiety B, single-domain ormulti-domain proteins, are covalently fused to the N-terminal ends of aFab fragment through linker sequences that are either cleavable ornon-cleavable by proteases. Fusion moiety A and fusion moiety B can haveantigen-specific binding capability or have no antigen specific bindingcapability.

FIG. 2. Schematic diagram depicting different conformations of thefusion moieties in MSFP and the relative strength of Fab bindingaffinities to cell surface target or soluble form of the target. Thelower panel of the figure shows the conformational flexibility of thefusion moieties in MSFP.

FIG. 3. Schematic diagrams illustrating the binding properties of Fab indifferent fusion formats to soluble and cell surface targets. Panel A,the structures of a Fab, a fusion protein of Fab with a single fusionmoiety at one of the N-termini, and a fusion protein of Fab with fusionmoieties at both N-termini of the Fab; Panel B. Schematic diagramsdepicting the Fab in all three Fab fusion formats capable of binding toits soluble target; Panel C. Schematic diagrams depicting the Fab domainwithout fusion and with single fusion moiety at the N-terminus of Fabcan bind to its target on cell surface, However, a Fab domain withfusions at the N-termini of both H- and L-chain can no longer bind toits target presented on cell surface due to steric hindrance.

FIG. 4. Schematic diagram depicting the necessary condition for a MSFPbinding to T cells: Panel A, MSFP with anti-tumor cell surface targetbinding moieties at the N-termini of both H- and L-chain can bind to atumor cell through tumor associated antigen (TAA) but does not bind toCD3 on a T cell (because of the dramatically reduced affinity, see FIG.3 Panel C); Panel B. When MSFP molecules with low affinity binding toCD3 on T cells can effectively engage tumor and T cells by avidityeffects. Representative MSFP molecules are clustered on tumor cellthrough TAA binding, the CD3 binding arms will bind to a T cell viaavidity, resulting in bridging a tumor cell and a T cell.

FIG. 5. Schematic diagram depicting a Fab fusion with single anti-tumorcell surface target moiety (or a single arm cleavage product from a Fabfusion at both H- and L-chain) can bind to targets both on tumor and Tcell resulting in bridging the two cell types.

FIG. 6. Panel A, Schematic diagram of Fabe showing two single chain Fv(scFv) domains as fusion moieties. A Fabe refers to a MSFP wherein theFab binding target is an immune cell surface effector molecules, such asCD3 epsilon chain or T cell receptor on T cells, NKG2D on NK cells, orFc gamma receptor on NK, monocytic cells and macrophage. The two scFvdomains can have the same specificity or different specificities; PanelB, schematic diagram of protein domains and the inter-chain disulfidebond in primary sequences. The inter chain disulfide bond between the CHand CL is optional. The configuration of the scFv can be in either VH-VLor VL-VH and the VH and VL configuration in scFv1 and scFv2 can be thesame or different.

FIG. 7. Panel A, Schematic diagram of Fabe-albu showing two single chainFv (scFv) domains as fusion moieties. A Fabe-albu is a specific type ofFabe where one scFv has binding specificity to human albumin and thesecond scFv has binding specificity to tumor cell surface antigen (TAA);Panel B, schematic diagram of protein domains and inter-chain disulfidebond in primary sequences. The configuration of the scFv can be ineither VH-VL or VL-VH and the VH and VL configuration in scFv1 and scFv2can be the same or different.

FIG. 8. 4-20% tris glycine SDS-PAGE (polyacrylamide gelanalysis)(Invitrogen) of mouse anti human CD3 antibody 1F3 and ahumanized 1F3 antibody, hu-1 F3. Gel was stained using SimplyBlue™reagent (Invitrogen). “NR” is under non reducing and “R” is underreducing condition with 5% mercaptoethanol. Protein bands indicate bothantibodies have normal sizes of intact IgG, IgGHC and IgGLC.

FIG. 9. Detection of reduced and denatured recombinant CD3 relatedproteins by mouse anti CD3 antibody, mu-1F3 and the humanized anti CD3antibody, hu-1 F3. 100 ng of indicated antigens were run on a 4-20% trisglycine gel and transferred to a nitrocellulose membrane. 1 μg/ml ofbiotinylated mu-1 F3 IgG (A) and biotinylated hu-1 F3 IgG was used toincubate with the membrane followed by streptavidin/HRP incubation.Detection was by ECL reagent. Fc(K-H) represents “knob and hole”heterodimerizing Fc mutants. CD3epsilonAA1-27.Fc is a CD3epsilonN-terminal peptide fusion with human Fc (see SEQ ID NO:18). The controlpeptide has the same amino acid composition as the CD3epsilonAA1-27 butwith reverse sequence order of the first 16 aa (see SEQ ID NO:20). Themouse anti CD3 IgG was able to recognize the denature CD3epsilon chainof human and cynomolgus species as well as the N-terminal amino acid1-27 of human CD3epsilon. A peptide with reverse order of amino acidsequence for the first 16 residues was not recognized by mouse 1F3 IgGsuggesting the positive bands are specific. Humanized 1 F3 antibody,hu-1 F3 IgG, showed very similar pattern of recognition of the samepanel of antigens indicating that this antibody has similar specificityand epitope as the parental mouse 1F3 antibody.

FIG. 10: Flow cytometric analysis of the binding to human PBMCs by mouseanti human CD3 antibody, mu-1F3 and its humanized version, hu-1F3. FACSstaining was done using biotinylated IgGs followed by a second stepusing streptavidin-PE conjugate. The mu-1F3 and the hu-1F3 IgG generatedsimilar cell staining pattern: large shift for T cells (2-3 logs), themajority of cells in human PBMCs. It is known that mouse Fc (in mu-1F3)lacks human Fcgamma receptor binding activity. Accordingly, the slightdifference in the low shifting cell population (0-1 log) is likely dueto the low affinity binding of hu-1F3 IgG (human IgG2 Fc) to the Fcgammareceptor expressing cells in the PBMC preparation. FACS was done using aFACSCALIBUR™ instrument (BD Bioscience).

FIG. 11: ELISA binding studies of mu-1F3 and hu-1F3 IgG to recombinantCD3epsilon related proteins. 50 μl of 1 μg/ml of indicated proteinantigens were coated on MAXISORP® ELISA plates (per well). Biotinylatedmu-1F3 IgG and hu-1F3 IgG (0.5 μg/ml in 4% milk TBS-0.05% TWEEN® 20(i.e., polysorbate 20)) were added to bind the immobilized antigensfollowed by streptavidin/HRP conjugate. ABTS™ (i.e.,2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) was used forcolor development and absorbance measured at 405 nm. ELISA on a controlpeptide Fc fusion (see SEQ ID NO: 20) are negative for both antibodies(data not shown). The results indicated very similar binding patternstowards different antigen forms by the parental mouse antibody, mu-1F3and the humanized version, hu-1F3 suggesting the specificity and theepitope for the humanized antibody are retained.

FIG. 12: Humanized 1F3 antibodies in Fab format recognize CD3 on Jurkatcells. His-tagged Fab proteins at concentration of 2 μg/ml wereincubated with Jurkat cells followed by mouse anti his-tag antibodyfollowed by anti-mouse PE-conjugate. FACS was run on a FACSCALBUR™instrument (BD biosciences).

FIG. 13: ELISA binding of humanized antiCD3 antibodies to recombinanthuman CD3epsilon/delta Fc(K-H) heterodimer (panels A and B); recombinanthuman CD3epsilon N-terminal amino acid 1-27 peptide Fc fusion (panels Cand D); recombinant cyno CD3epsilon/delta Fc(K+L) heterodimer (panels Eand F). Humanized Fab proteins are his6 tagged at the C-terminus of Fdand the Fabs bound to the 96-well plate immobilized antigens weredetected using anti his6 tag mouse antibody conjugated with HRP. ABTSwas used as substrate and absorptions were measured at 405 nm.

FIG. 14: EpCAM×CD3 Fab fusion proteins (MSFP) binding to Jurkat cellsexpressing human CD3 using FACS analysis. Biotinylated Fab and Fabfusions bound to Jurkats were detected by streptavidin-PE conjugate.OKT3 Fab showed binding to CD3 on Jurkats but the anti EpCAM scFv fusionto either the LC alone or to both HC and LC completely abolished the CD3binding ability of OKT3 Fab moiety. Humanized anti CD3 antibody Fab,hu-1 F3 is able to bind CD3 on Jurkat cells. Contrary to OKT3 Fab fusionproteins, anti EpCAM scFv fused to the N-terminal of hu-1F3 LC retainedthe binding activity at similar level as the hu-1F3 Fab; simultaneousfusion of anti EpCAM scFv fusion to the LC and HC of hu-1F3 Fab showedpositive binding to CD3 on Jurkat cells but at a reduced level.

FIG. 15: EpCAM×CD3 MSFP binding to human PBMCs using FACS analysis.Biotinylated Fab and Fab fusions bound to Jurkat cells were detected bystreptavidin-PE conjugate. Panel A) OKT3 Fab bound to PBMCs expressingCD3 (T cells); the anti EpCAM scFv fused to both HC and LC completelyabolished the CD3 binding ability of OKT3 Fab moiety. Panel B) HumanizedantiCD3 antibody Fab, hu-1F3.1 is able to bind CD3 on T cells in PBMCpreparation; unlike the OKT3 Fab fusion protein, anti EpCAM×hu-1F3.1 Fabwith simultaneous fusions to the LC and HC of Fab showed positivebinding to CD3 on Jurkat cells and the level of binding is at a reducedlevel.

FIG. 16: ELISA binding assay showed that: Panel A) OKT3 Fab andEpCAM1.1×OKT3 bispecific Fab fusion proteins have no binding activitytowards the recombinant cyno CD3epsilon/delta heterodimeric Fc protein;and Panel B) OKT3 Fab and bispecific fusions have no binding activitytowards the recombinant human CD3epsilon N-terminal peptide (aa1-27.Fcfusion). Fab and Fab fusion proteins were biotinylated and the-biotinylated antibodies bound to antigens immobilized on 96-well platewere detected using streptavidin-HRP conjugate followed by ABTSsubstrate for detection.

FIG. 17: ELISA binding assay showed that hu-1F3 Fab andEpCAM1.1×hu-1F3.1 bispecific Fab fusion proteins (MSFP; Fabe) havepositive binding activities Panel A) towards the recombinant cynoCD3epsilon/delta Fc(K+H) heterodimeric protein; and Panel B) towards therecombinant human CD3epsilon N-terminal peptide (aa1-27.Fc fusion). Faband Fab fusion proteins were biotinylated and the biotin-antibodiesbound to 96-well plate immobilized antigens were detected usingstreptavidin-HRP conjugate followed by ABTS as substrate for detection.

FIG. 18: ELISA binding assay showed that hu-1F3 Fab andEpCAM1.2×hu-1F3.1 MSFP have positive binding activities Panel A) towardsthe recombinant cyno CD3epsilon/delta Fc(K+H) heterodimeric protein; andPanel B) towards the recombinant human CD3epsilon N-terminal peptide(aa1-27.Fc fusion). Fab and Fab fusion proteins were biotinylated andthe labeled fusion proteins bound to 96-well plate immobilized antigenswere detected using streptavidin-HRP conjugate followed by ABTS assubstrate for detection.

FIG. 19: ELISA binding assay showed that hu-1F3.1 Fab andEpCAM2.2×hu-1F3 MSFP have positive binding activities Panel A) towardsthe recombinant cyno CD3epsilon/delta Fc(K+H) heterodimeric protein; andPanel B) towards the recombinant human CD3epsilon N-terminal peptide(aa1-27.Fc fusion). Fab and Fab fusion proteins were biotinylated andthe biotin-labeled fusion proteins bound to 96-well plate immobilizedantigens were detected using streptavidin-HRP conjugate followed by ABTSsubstrate for detection.

FIG. 20: EpCAM×CD3 MSFP binding to CHO cells stably expressing fulllength human EpCAM (hu-EpCAM-FL) target on cell surface by FACSanalysis. Anti-CD3 Fab has no binding to the same CHO cells as expected.EpCAM×CD3 MSFPs show positive binding to EpCAM expressed on CHO cells.

FIG. 21: FACS based assay to detect PBMC (T cells) mediated cell killingactivity redirected by EpCAM×CD3 MSFP in a tumor target dependentmanner. In this assay, target cells (CHO cells stably expressing humanEpCAM-FL on cell surface (Panels A, B, and D) and control cells (CHOonly, Panel C) were labeled with PKH-26 fluorescence dye (Sigma,according to product instruction) prior to assay. TOPRO®-3 (TP3,Invitrogen, i.e. a carbocyanine monomer nucleic acid stain) was used toidentify dead cells at the end of the cell killing assay. In this assay,dead target cells were identified from the counts in the upper-rightquadrant (PKH-26 positive, TP3 positive) and the live target cells wereidentified from the upper-left quadrant (PKH-26 positive, TP3 negative).Panel A) EpCAM expressing CHO cells incubated (for 20 hrs) withEpCAM2.2×CD3 MSFP but without PBMCs had ˜1.4% dead cells (PKH-26labelled TP3 positive cells); Panel B) EpCAM-expressing CHO cellincubated (for 20 hrs) with PBMCs but without MSFP had ˜14% dead cellsamong the PKH-26 labelled populations (non-specific killing activity ofPBMC; Panel C) non EpCAM-expressing CHO cells incubated (for 20 hrs)with PBMCs and EpCAM2.2×CD3 MSFP had a ˜14% dead cells (non-specifickilling activity of PBMCs); and Panel D) EpCAM-expressing CHO cellsincubated (for 20 hrs) with PBMCs and exemplary EpCAM×CD3 MSFP had ˜64%dead cell counts. The tumor cell target dependent % killing activity ofEpCAM2.2×CD3 MSFP is ˜50% (comparing Panels C and D or B and D).

FIG. 22: The redirected T cell cytolytic activity of MSFPs towards theEpCAM-expressing CHO cells using the FACS assay described in FIG. 18.Panel A) OKT3 Fab and its MSFPs lack significant redirected cytolyticactivity; While hu-1 F3.1 Fab had no activity towards target cells,Panel B) EpCAM1.1×hu-1 F3.1 MSFP had high level activity and theactivity level remained high even at 60 pM MSFPs; Panel C) dosedependent activities were detected by EpCAM1.2×hu-1 F3.1 MSFP; and PanelD) dose dependent cytolytic activities were detected for EpCAM2.2 scFvsingle fusions to hu-1 F3.1 Fab. For double fusion of EpCAM2.2 scFv toboth the HC and LC of hu-1F3.1 Fab, a maximum killing of 50% cellpopulation were observed and the activity level remained high (˜40%)when its concentration was at as low as 60 pM. Percentage killingactivities were calculated by subtracting percentage of dead cells inthe control assay (EpCAM-CHO+PBMC+no MSFP) from the percentage of deadcells in the sample assay.

FIG. 23: T cell activation (in PBMC) by EpCAM2.2-(H+L)-hu-1F3.1 MSFP istumor target dependent. Panel A) PBMCs incubated withEpCAM2.2-(H+L)-hu-1F3.1 (30 nM) in the presence of non EpCAM-expressingCHO resulted in basal level T cell activation measured by CD69expression by FACS analysis; Panel B) PBMCs incubated withEpCAM2.2-(H+L)-hu-1F3.1 (30 nM, 16 hrs) in the presence of nonEpCAM-expressing CHO resulted in significant increase of T cellactivation measured by CD69 expression detected by FACS assay.

DETAILED DESCRIPTION

The present disclosure relates to multi-specific Fab fusion proteins(MSFP). In particular, the MSFP of the present disclosure comprise a Fabfragment which binds a particular target antigen of interest (e.g., CD3,T cell receptor, NKG2D, or FcγR) and has one or two fusion moietieswhich bind to one or, in some embodiments, two additional targetantigens (e.g., a serum albumin protein, a tumor antigen or otherdisease-associate antigen).

It is envisioned that the effectiveness of cancer treatment includingthe efficacy, safety, costs related to the manufacturing of drugsubstance and route of drug administration can be dramatically improvedby developing innovative drugs possessing most or all of the followingattributes:

1. Enhanced selectivity for targeting tumor cells over normal cellsusing bivalent tumor associated antigen (TAA) binding molecules. Theenergetics of antibody and antigen interaction is usually expressed bythe terms of affinity and avidity. Avidity is distinct from affinity,which is a term used to describe the strength of a single siteinteraction between an antibody binding domain and an antigen. As such,avidity is the combined synergistic strength of affinities. Antibodybinding to tumor cells is dependent on the intrinsic binding affinity,the number of binding sites present on an antibody (valency) and thedensity of the target antigen on cells (Reynolds, Biochemistry 18:264-9,1979). When a monovalent antibody is used, the measured binding strengthis related to affinity. When a bivalent or multivalent antibody is usedfor cell binding, avidity is usually measured. Higher antigenconcentration usually leads to higher avidity. It is known that manytumor cells overexpress TAA on the tumor cell surface. MSFP with twoanti-tumor target binding domains can have selectively higher binding totumor cells over-expressing TAA than normal tissue cells also expressingthe same target. Based on the avidity principle, Adams et al.successfully demonstrated that using a bivalent anti her2/neu antibodyfragment improved the accumulation in tumor mass 3 times better thanusing a corresponding monovalent version of the same antibody fragment(Adams et al., Clin Cancer Res 12:1599-1605, 2006). In terms of tumortargeting, high affinity of antibody usually results in the accumulationat the periphery of the tumor mass while high avidity effect leads todeeper penetration of the antibody into the tumors.

2. Enhanced selectivity at targeting tumor over normal cells usingbispecific antibodies. Bispecific reagents can be designed for theirgoals to bind and kill tumor cells more selectively over normal cellsand ultimately to increase the efficacy and safety over a monospecificreagent (Chang et al., Mol Cancer Ther 1:553, 2002; Kipriyanov and LeGall, Curr Opin Drug Discov Devel 7:233, 2004). Requirement for thepresence of both antigens on tumor cells and careful design of properaffinities of each antigen binding arm are critical aspects of thisapproach. For example, MM-111 is an anti Her2 and Her3 bispecific fusionprotein designed to use her2 arm to target the drug to cancer cells andto use anti her3 arm to inhibit the her3 receptor heterodimerization andsignaling in cancer cells (Robinson et al., British Journal of Cancer99, 1415-1425, 2008). Important aspects considered for the design ofMM-111 include: 1) relatively restricted tissue expression pattern andcancer cell overexpression of Her2, 2) broad tissue expression of Her3and critical importance of Her3 signalling in cancer cell growth andsurvival, 3) high affinity binding to Her2 (1.1 nM) and low affinitybinding to Her3 (160 nM). In an in vitro binding assay, MM-111demonstrated up to 9.7 fold high binding to Her2/Her3 doubly positivecells than Her2 single positive cells while there was no detectiblebinding to Her3 single positive cells at a concentration as high as 1 μM(Robinson et al., British Journal of Cancer 99, 1415-1425, 2008).

Thus, the present disclosure provides these and other advantages asdescribed further herein. In particular, the MSFP of the presentdisclosure provide distinct advantages, including but not limited to thefollowing: 1) The MSFP do not bind or have reduced binding to theprimary Fab target when the Fab is bound to fusion moieties. E.g., anMSFP comprising an anti-CD3 Fab does not bind to or has significantlyreduced binding to CD3 when the Fab is bound to fusion moiety A andfusion moiety B, particularly when the fusion moieties are not engagedwith their target. This lowers unwanted side-effects (e.g., unwantedgeneral activation of T cells prior to binding tumor cells) or othercells expressing Fab target antigen). 2) Bivalent binding or bispecificbinding enhances the selectivity of the MSFP for tumor versus normalcells. 3) The MSFP is relatively large (˜75-100 kD) thereby avoidingfast renal clearance (filtration threshold size for renal clearance is70 kD) but is smaller than a regular antibody (150 kD) which allowsimproved tumor tissue penetration. 5) The ability of some MSFP to bindserum albumin dramatically enhances the circulation half-life. 6) TheMSFP structure tends to be more stable than fusion proteins having onlyscFv.

In certain embodiments, the MSFPs of the present disclosure are bivalentwith respect to binding a specific target antigen (e.g., tumor antigen)and are bispecific in that it binds a target tumor antigen via thefusion moieties and a second target antigen via the Fab binding domain(e.g., CD3, T cell receptor, NKG2D, or FcγR) which enhances its efficacythough activation of, for example, the immune system against cellsexpressing a particular target antigen.

The illustrative MSFP of the present disclosure comprises an anti-CD3Fab. However, other Fab targets are specifically contemplated herein.Concerning the anti-CD3 Fab, to date, protein molecules directed againstthe TCR complex either, together with T cell activation, also induce a Tcell signal resulting in a cytokine storm, which can result in severeside effects, or have little effect on cells in the absence ofcross-linking. The present MSFP provides the advantage of inhibiting TCR(CD3) binding and activation unless the MSFP first binds to a secondarytarget antigen, e.g., a tumor antigen, thus activating T cells. Thisgreatly reduces unwanted cytokine storm and unwanted activation of Tcells in the absence of the desired target cell (e.g., a tumor celltarget).

The following description is merely intended to illustrate variousembodiments of the present disclosure. As such, the specificmodifications discussed are not intended to be limiting. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the spirit or scopeof the subject matters presented herein, and it is understood that suchequivalent embodiments are to be included herein.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Current Protocols in MolecularBiology or Current Protocols in Immunology, John Wiley & Sons, New York,N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology,3^(rd) ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning:A Laboratory Manual (3rd Edition, 2001); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984) and other like references.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturers specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

“C-terminal” of a polypeptide as used herein refers to the last aminoacid residue of the polypeptide which donates its amine group to form apeptide bond with the carboxyl group of its adjacent amino acid residue.“N-terminal” of a polypeptide as used herein refers to the first aminoacid of the polypeptide which donates its carboxyl group to form apeptide bond with the amine group of its adjacent amino acid residue.

The term “covalently link” as used herein refers to direct linkingthrough one or more chemical bonds or indirect linking through one ormore linkers.

Any suitable chemical bonds can be used to make a direct link, includingwithout limitation, covalent bonds such as peptide bond and disulfidebond, non-covalent bonds such as hydrogen bond, hydrophobic bond, ionicbond, and Van der Waals bond.

A “covalent bond” refers herein to a stable association between twoatoms which share one or more electrons. Examples of the covalent bondsinclude, without limitation, a peptide bond and a disulfide bond.“Peptide bond” as used herein refers to the covalent bond formed betweenthe carboxyl group of an amino acid and the amine group of the adjacentamino acid. “Disulfide bond” as used herein refers to a covalent bondformed between two sulfur atoms. A disulfide bond can be formed fromoxidation of two thiol groups. In certain embodiments, the covalentlylink is direct link through a covalent bond. In certain embodiments, thecovalently link is direct link through a peptide bond or a disulfidebond.

A “non-covalent bond” refers herein to an attractive interaction betweentwo molecules or two chemical groups that does not involve sharing ofelectrons. Examples of non-covalent bonds include, without limitation, ahydrogen bond, a hydrophobic bond, an ionic bond, and a Van der Waalsbond. A “hydrogen bond” refers herein to attractive force between ahydrogen atom of a first molecule/group and an electronegative atom of asecond molecule/group. A “hydrophobic bond” refers herein to a forcethat causes hydrophobic or non-polar molecules/groups to aggregate orassociate together in an aqueous environment. An “ionic bond” refersherein to an attraction between a positive ion and a negative ion. A“Van der Waals bond” refers herein to a non-specific attraction forcebetween two adjacent molecules/groups which have momentary randomfluctuations in the distribution of electrons. In certain embodiments,the covalently link is direct link through a non-covalent bond. Incertain embodiments, the covalently link is direct link through ahydrogen bond, a hydrophobic bond, an ionic bond, or a Van der Waalsbond.

A binding domain (or a fusion protein thereof) “specifically binds” to atarget molecule if it binds to or associates with a target molecule withan affinity or Ka (i.e., an equilibrium association constant of aparticular binding interaction with units of 1/M) of, for example,greater than or equal to about 10⁵ M⁻¹. In certain embodiments, abinding domain (or a fusion protein thereof) binds to a target with a Kagreater than or equal to about 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or 10¹³ M⁻¹. “High affinity” binding domains(or single chain fusion proteins thereof) refers to those bindingdomains with a K_(a) of at least 10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 10⁹M⁻¹, at least 10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 10¹² M⁻¹, at least10¹³ M⁻¹, or greater. Alternatively, affinity may be defined as anequilibrium dissociation constant (K_(d)) of a particular bindinginteraction with units of M (e.g., 10⁻⁵ M to 10⁻¹³ M, or less).Affinities of binding domain polypeptides and fusion proteins accordingto the present disclosure can be readily determined using conventionaltechniques (see, e.g., Scatchard et al. (1949) Ann. N.Y. Acad. Sci.51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614, or the equivalent).

“Derivative” as used herein refers to a chemically or biologicallymodified version of a compound (e.g., a protein) that is structurallysimilar to a parent compound and (actually or theoretically) derivablefrom that parent compound.

The term “steric hindrance” refers to the prevention or retardation of abinding interaction between molecules, resulting from their sizes orspatial disposition.

The term “avidity” is a term used to describe the combined strength ofmultiple bond interactions. Avidity is distinct from affinity, which isa term used to describe the strength of a single bond. With regard toantibodies, avidity refers to antibody interactions in which multipleantigen binding sites simultaneously interact with targets.Individually, each binding interaction may be readily broken, however,when many binding interactions are present at the same time, transientunbinding of a single site does not allow the molecule to diffuse away,and binding of that site is likely to be reinstated. The overall effectis synergistic, strong binding of antigen to antibody.

Multi-Specific Fab Fusion Proteins

The present disclosure provides multi-specific Fab fusion proteins(MSFP; also referred to in the figures as Fabe where the Fab binds to animmune effector molecule, such as CD3 epsilon chain, T cell receptor,NKG2D, or FcγR) that comprise a Fab fragment (e.g., of the basicstructure NH₂-VL-CL-S-S-CH1-VH-NH₂) having attached thereto a firstfusion moiety at the N-terminal end of the VL and/or a second fusionmoiety attached at the N-terminal end of the VH. Between the fusionmoieties and the VH and VL there may be a linker which is optionallyprotease cleavable. This general format is the basic structure that canbe built upon to construct more complex homodimer multi-specific Fabfusion protein complexes depending on the fusion moieties used asdescribed further below.

Prior to the present application, there has been no description for aFab-based fusion having fusion moieties at the N-terminal end of boththe heavy chain and the light chain of the Fab. As would be understoodby the skilled person, such fusions would severely cripple Fab bindingaffinity thus to render them practically non-useful. This general notionof reduced binding upon fusion of binding domain(s) to the N-terminus ofa Fab was observed in MSFP. For example, fusion to the N-terminus of theOKT3 anti-CD3 Fab significantly reduced the binding to the Fab targetand resulted in almost complete loss of biological activity of thebispecific Fab fusion proteins. However it is surprising that fusion ofantigen binding domains to the N-terminus of the hu-1 F3.1 humanizedanti-CD3 Fab, in some cases did not result in significant loss ofbinding to the Fab target. In some cases, loss of binding to cellsurface target was more significant than to the soluble protein target.In some other cases, loss of binding did not result in loss ofbiological activity. On the contrary, MSFP with lower binding affinityto CD3 on T cells can have higher biological activity. While this issurprising, it underscores the importance of at least, but not limitedto, two factors: 1) upon high binding to tumor cells (using the bindingdomains of fusion moieties A and B), MSFP exhibit high avidity toovercome the low affinity of the Fab portion for binding to T cells,thus inducing killing; 2) It is highly antibody-, thereforeepitope-dependent because OKT3 Fab fusion proteins behaved completelydifferently than the anti CD3 antibody, 1F3 derived MSFPs. The MSFP inthis disclosure made with Fab fragments derived from antibodies bindingto the CD3 epsilon epitope recognized by 1F3 and its humanized variants,possesses many unique features and these features can be utilized todevelop human therapeutics with desirable attributes in drug safety,efficacy and manufacturability. As described herein, this property isused advantageously in the MSFP of the present disclosure to mask Fabbinding until the MSFP are in an appropriate environment (e.g., in thevicinity of a tumor).

The specific structural components of the MSFP are described in moredetail in the sections below. The function of the MSFP is described inmore detail in the section below entitled “Function of theMulti-Specific Fab Fusion Proteins”.

Fab Fragment

As noted above, the multi-specific Fab fusion proteins disclosed hereincomprise at their core, a Fab fragment. As would be understood by theskilled person, a Fab fragment is the antigen-binding fragment of anantibody. The Fab is composed of one constant and one variable region ofan immunoglobulin heavy and an immunoglobulin light chain. The heavychain constant and variable regions heterodimerize with the light chainvariable and constant regions and are usually covalently linked by adisulfide bond between the heavy and light chain constant regions (seee.g., diagram in FIG. 1). Thus, as used herein, “Fab” with regard to anantibody generally refers to that portion of the antibody consisting ofa single light chain (both variable and constant regions) bound to thevariable region and first constant region of a single heavy chain by adisulfide bond.

As would be recognized by the skilled person, a disulfide bond betweenthe heavy and light chain is preferable, but not essential for function(Orcutt et al. (2010), PEDS, 23:221-228). Thus, in certain embodimentsthe Fab fragment of the present invention may not comprise a disulfidebond. In this regard, the heavy and light chains may be engineered insuch a way so as to stably interact without the need for disulfide bond.For example, in certain embodiments, the heavy or light chain can beengineered to remove a cysteine residue and wherein the heavy and lightchains still stably interact and function as a Fab. In one embodiment,mutations are made to facilitate stable interaction between the heavyand light chains. For example, a “knobs into holes” engineering strategycan be used to facilitate dimerization between the heavy and lightchains of a Fab (see e.g., 1996 Protein Engineering, 9:617-621). Usingthis strategy, “knobs” are created by replacing small amino acid sidechains at the interface between interacting domains with larger ones.Corresponding “holes” are made at the interface between interactingmolecules by replacing large side chains with smaller ones. Thus, alsocontemplated for use herein are variant Fab fragments designed for aparticular purpose, for example, amino acid changes in the constantdomains of CH1 and or CL, and removal of a disulfide bond or addition oftags for purification, etc.

In another embodiment, the configuration of the variable and constantregions within the Fab fragment may be different from what is found in anative Fab. In other words, in one embodiment, the orientation of thevariable and constant regions may be VH-CL in one chain and in anotherVL-CH1 (Schaefer et al. (2011), PNAS, 108:111870-92). Such modified Fabfragments still function to bind their particular target antigen and arecontemplated for use in the MSFPs of the present invention. Thus, inthis regard the variable regions and constant regions that make up theFab are considered modular.

In certain embodiments, the Fab fragments of this disclosure are derivedfrom monoclonal antibodies and may be derived from antibodies of anytype, including IgA, IgM, IgD, IgG, IgE and subtypes thereof, such asIgG1, IgG2, IgG3, and IgG4. The light chain domains may be derived fromthe kappa or lambda chain. The Fab fragments for use herein may be maderecombinantly.

As is well known in the art, an antibody is an immunoglobulin moleculecapable of specific binding to a target, such as a carbohydrate,polynucleotide, lipid, polypeptide, etc., through at least one epitoperecognition site, located in the variable region of the immunoglobulinmolecule. As used herein, the term encompasses not only intactpolyclonal or monoclonal antibodies, but also humanized antibodies,chimeric antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen-binding site orfragment (epitope recognition site) of the required specificity.

The Fab fragment as disclosed herein comprises an antigen-bindingportion comprised of an immunoglobulin heavy chain variable region andan immunoglobulin light chain variable region (VH and VL). Morespecifically, the term “antigen-binding portion” as used herein refersto a polypeptide fragment that contains at least one CDR of animmunoglobulin heavy and/or light chains that binds to the targetantigen of interest, such as the CD3 molecule. In this regard, anantigen-binding portion of the herein described multi-specific Fabfusion proteins may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VLsequence of a parent antibody that binds to a target antigen ofinterest. In certain embodiments, the antigen-binding portion of the Fabfragment of an MSFP binds to CD3.

In certain embodiments, a specific VH and/or VL of the MSFP describedherein may be used to screen a library of the complementary variableregion to identify VH/VL with desirable properties, such as increasedaffinity for a target antigen of interest. Such methods are described,for example, in Portolano et al., J. Immunol. (1993) 150:880-887;Clarkson et al., Nature (1991) 352:624-628.

Other methods may also be used to mix and match CDRs to identify Fabhaving desired binding activity (such as binding to CD3, or other targetantigen of interest as described herein for other binding domainspresent in the fusion moieties of the MSFP). For example: Klimka et al.,British Journal of Cancer (2000) 83: 252-260, describe a screeningprocess using a mouse VL and a human VH library with CDR3 and FR4retained from the mouse VH. After obtaining antibodies, the VH wasscreened against a human VL library to obtain antibodies that boundantigen. Beiboer et al., J. Mol. Biol. (2000) 296:833-849 describe ascreening process using an entire mouse heavy chain and a human lightchain library. After obtaining antibodies, one VL was combined with ahuman VH library with the CDR3 of the mouse retained. Antibodies capableof binding antigen were obtained. Rader et al., PNAS (1998) 95:8910-8915describe a process similar to Beiboer et al above.

These just-described techniques are, in and of themselves, known as suchin the art. The skilled person will, however, be able to use suchtechniques to obtain antigen-binding fragments of antibodies accordingto several embodiments of the disclosure described herein, using routinemethodology in the art.

Also disclosed herein is a method for obtaining an antibody antigenbinding domain specific for a target antigen (e.g., CD3 or any targetantigen described elsewhere herein for targets of fusion moiety bindingdomains), the method comprising providing by way of addition, deletion,substitution or insertion of one or more amino acids in the amino acidsequence of a VH domain set out herein a VH domain which is an aminoacid sequence variant of the VH domain, optionally combining the VHdomain thus provided with one or more VL domains, and testing the VHdomain or VH/VL combination or combinations to identify a specificbinding member or an antibody antigen binding domain specific for atarget antigen of interest (e.g., CD3) and optionally with one or moredesired properties. The VL domains may have an amino acid sequence whichis substantially as set out herein. An analogous method may be employedin which one or more sequence variants of a VL domain disclosed hereinare combined with one or more VH domains.

An epitope that “specifically binds” or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances.

An antibody, or Fab or scFv thereof, “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to a CD3 epitope is an antibody that binds one CD3epitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other CD3 epitopes or non-CD3epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

Immunological binding generally refers to the non-covalent interactionsof the type which occur between an immunoglobulin molecule and anantigen for which the immunoglobulin is specific, for example by way ofillustration and not limitation, as a result of electrostatic, ionic,hydrophilic and/or hydrophobic attractions or repulsion, steric forces,hydrogen bonding, van der Waals forces, and other interactions. Thestrength, or affinity of immunological binding interactions can beexpressed in terms of the dissociation constant (K_(d)) of theinteraction, wherein a smaller K_(D) represents a greater affinity.Immunological binding properties of selected polypeptides can bequantified using methods well known in the art. One such method entailsmeasuring the rates of antigen-binding site/antigen complex formationand dissociation, wherein those rates depend on the concentrations ofthe complex partners, the affinity of the interaction, and on geometricparameters that equally influence the rate in both directions. Thus,both the “on rate constant” (k_(d)) can be determined by calculation ofthe concentrations and the actual rates of association and the “off rateconstant” (k_(off)) and can be determined by the actual rates ofdissociation. The ratio of k_(off)/k_(on) is thus equal to thedissociation constant K_(D). See, generally, Davies et al. (1990) AnnualRev. Biochem. 59:439-473.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantigen-binding portion of a Fab fragment, and additionally capable ofbeing used in an animal to produce antibodies capable of binding to anepitope of that antigen. An antigen may have one or more epitopes.

The term “epitope” includes any determinant, in certain embodiments, apolypeptide determinant, capable of specific binding to animmunoglobulin or T-cell receptor. An epitope is a region of an antigenthat is bound by an antibody or an antigen-binding fragment thereof. Incertain embodiments, epitope determinants include chemically activesurface groupings of molecules such as amino acids, sugar side chains,phosphoryl or sulfonyl, and may in certain embodiments have specificthree-dimensional structural characteristics, and/or specific chargecharacteristics. In certain embodiments, an MSFP is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules. An MSFP is said tospecifically bind an antigen when the equilibrium dissociation constantis ≤10⁻⁵, 10⁻⁶ or 10⁻⁷ M. In some embodiments, the equilibriumdissociation constant may be ≤10⁻⁸ M or ≤10⁻⁹ M. In some furtherembodiments, the equilibrium dissociation constant may be ≤10⁻¹⁰ M or≤10⁻¹¹ M

In certain embodiments, antigen-binding portions of the Fab fragment asdescribed herein include a heavy chain and a light chain CDR set,respectively interposed between a heavy chain and a light chainframework region (FR) set which provide support to the CDRs and definethe spatial relationship of the CDRs relative to each other. As usedherein, the term “CDR set” refers to the three hypervariable regions ofa heavy or light chain V region. Proceeding from the N-terminus of aheavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and“CDR3” respectively. An antigen-binding site, therefore, includes sixCDRs, comprising the CDR set from each of a heavy and a light chain Vregion. A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 orCDR3) is referred to herein as a “molecular recognition unit.”Crystallographic analysis of a number of antigen-antibody complexes hasdemonstrated that the amino acid residues of CDRs form extensive contactwith bound antigen, wherein the most extensive antigen contact is withthe heavy chain CDR3. Thus, the molecular recognition units areprimarily responsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRs. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

The structures and locations of immunoglobulin variable regions may bedetermined by reference to Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest. 4th Edition. US Department of Health and HumanServices. 1987, and updates thereof, now available on the Internet(immuno.bme.nwu.edu).

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an epitope. Monoclonal antibodies are highlyspecific, being directed against a single epitope. The term “monoclonalantibody” encompasses not only intact monoclonal antibodies andfull-length monoclonal antibodies, but also fragments thereof (such asFab, Fab′, F(ab′)₂, Fv), single chain (ScFv), variants thereof, fusionproteins comprising an antigen-binding portion, humanized monoclonalantibodies, chimeric monoclonal antibodies, and any other modifiedconfiguration of the immunoglobulin molecule that comprises anantigen-binding fragment (epitope recognition site) of the requiredspecificity and the ability to bind to an epitope. It is not intended tobe limited as regards the source of the antibody or the manner in whichit is made (e.g., by hybridoma, phage selection, recombinant expression,transgenic animals, etc.). The term includes whole immunoglobulins aswell as the fragments etc. described above under the definition of“antibody”.

The proteolytic enzyme papain preferentially cleaves IgG molecules toyield several fragments, two of which (the F(ab) fragments) eachcomprise a covalent heterodimer that includes an intact antigen-bindingsite. The enzyme pepsin is able to cleave IgG molecules to provideseveral fragments, including the F(ab′)₂ fragment which comprises bothantigen-binding sites. An Fv fragment for use according to certainembodiments of the present invention can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions of an IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H):V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

In one particular embodiment of the present disclosure, the Fab fragmentbinds to CD3. “T cell receptor” (TCR) is a molecule found on the surfaceof T cells that, along with CD3, is generally responsible forrecognizing antigens bound to major histocompatibility complex (MHC)molecules. It consists of a disulfide-linked heterodimer of the highlyvariable (alpha) and (beta) chains in most T cells. In other T cells, analternative receptor made up of variable Y and (delta) chains isexpressed. Each chain of the TCR is a member of the immunoglobulinsuperfamily and possesses one N-terminal immunoglobulin variable region,one immunoglobulin constant region, a transmembrane region, and a shortcytoplasmic tail at the C-terminal end (see, Abbas and Lichtman,Cellular and Molecular Immunology (5th Ed.), Editor: Saunders,Philadelphia, 2003; Janeway et al., Immunobiology: The Immune System inHealth and Disease, 4th Ed., Current Biology Publications, p 148, 149,and 172, 1999). TCR as used in the present disclosure may be fromvarious animal species, including human, mouse, rat, or other mammals.

“Anti-TCR Fab” or “Anti-TCR Fabe”, refers to a Fab or an MSFP comprisingsuch a Fab that specifically binds to a TCR molecule or one of itsindividual chains (e.g., TCR (alpha), TCR(beta), TCRY or TCR(delta)chain). In certain embodiments, an anti-TCR Fab binds to a TCR (alpha),a TCR(beta), or both.

“CD3” is known in the art as a multi-protein complex of six chains (see,Abbas and Lichtman, 2003; Janeway et al., p 172 and 178, 1999). Inmammals, the complex comprises a CD3(gamma) chain, a CD3(delta) chain,two CD3(epsilon) chains, and a homodimer of CD3(zeta) chains. TheCD3(gamma), CD3(delta), and CD3(epsilon) chains are highly related cellsurface proteins of the immunoglobulin superfamily containing a singleimmunoglobulin domain. The transmembrane regions of the CD3(gamma),CD3(delta), and CD3(epsilon) chains are negatively charged, which is acharacteristic that allows these chains to associate with the positivelycharged T cell receptor chains. The intracellular tails of theCD3(gamma), CD3(delta), and CD3(epsilon) chains each contain a singleconserved motif known as an immunoreceptor tyrosine-based activationmotif or ITAM, whereas each CD3(zeta) chain has three. Without wishingto be bound by theory, it is believed the ITAMs are important for thesignaling capacity of a TCR complex. CD3 as used in the presentdisclosure may be from various animal species, including human, mouse,rat, or other mammals.

“Anti-CD3 Fab” as used herein, refers to a Fab that specifically bindsto individual CD3 chains (e.g., CD3(gamma) chain, CD3(delta) chain,CD3(epsilon) chain) or a complex formed from two or more individual CD3chains (e.g., a complex of more than one CD3(epsilon) chains, a complexof a CD3(gamma) and CD3(epsilon) chain, a complex of a CD3(delta) andCD3(epsilon) chain). In certain embodiments, an anti-CD3 Fabspecifically binds to a CD3(gamma), a CD3(delta), a CD3(epsilon), or anycombination thereof, and in certain embodiments, a CD3(epsilon). In oneembodiment, an anti-CD3 Fab binds to the N-terminus of CD3 epsilon. Inone particular embodiment, the anti-CD3 Fab binds to amino acids 1-27 ofCD3 epsilon.

“TCR complex,” as used herein, refers to a complex formed by theassociation of CD3 with TCR. For example, a TCR complex can be composedof a CD3(gamma) chain, a CD3(delta) chain, two CD3(epsilon) chains, ahomodimer of CD3(zeta) chains, a TCR(alpha) chain, and a TCR(beta)chain. Alternatively, a TCR complex can be composed of a CD3(gamma)chain, a CD3(delta) chain, two CD3(epsilon) chains, a homodimer ofCD3(zeta) chains, a TCRY chain, and a TCR(delta) chain.

“A component of a TCR complex,” as used herein, refers to a TCR chainTCR(alpha), TCR(beta), TCRY or TCR(delta)), a CD3 chain (i.e.,CD3(gamma), CD3(delta), CD3(epsilon) or CD3(zeta)), or a complex formedby two or more TCR chains or CD3 chains (e.g., a complex of TCR(alpha)and TCR(beta), a complex of TCRY and TCR(delta), a complex ofCD3(epsilon) and CD3(delta), a complex of CD3(gamma) and CD3(epsilon),or a sub-TCR complex of TCR(alpha), TCR(beta), CD3(gamma), CD3(delta),and two CD3(epsilon) chains).

By way of background, the TCR complex is generally responsible forinitiating a T cell response to antigen bound to MHC molecules. It isbelieved that binding of a peptide:MHC ligand to the TCR and aco-receptor (i.e., CD4 or CD8) brings together the TCR complex, theco-receptor, and CD45 tyrosine phosphatase. This allows CD45 to removeinhibitory phosphate groups and thereby activate Lck and Fyn proteinkinases. Activation of these protein kinases leads to phosphorylation ofthe ITAM on the CD3(zeta) chains, which in turn renders these chainscapable of binding the cytosolic tyrosine kinase ZAP-70. The subsequentactivation of bound ZAP-70 by phosphorylation triggers three signalingpathways, two of which are initiated by the phosphorylation andactivation of PLC-(gamma), which then cleaves phosphatidylinositolphosphates (PIPs) into diacylglycerol (DAG) and inositol trisphosphate(IP3). Activation of protein kinase C by DAG leads to activation of thetranscription factor NFKB. The sudden increase in intracellular freeCa²⁺ as a result of IP3 action activates a cytoplasmic phosphatase,calcineurin, which enables the transcription factor NFAT (nuclear factorof activated T cells) to translocate form the cytoplasm to the nucleus.Full transcriptional activity of NFAT also requires a member of the AP-1family of transcription factors; dimers of members of the Fos and Junfamilies of transcription regulators.

A third signaling pathway initiated by activated ZAP-70 is theactivation of Ras and subsequent activation of a MAP kinase cascade.This culminates in the activation of Fos and hence of the AP-1transcription factors. Together, NFKB, NFAT, and AP-1 act on the T cellchromosomes, initiating new gene transcription that results in thedifferentiation, proliferation and effector actions of T cells. See,Janeway et al., p 178, 1999.

In certain embodiments, the Fab specifically binds to an individualhuman CD3 chain (e.g., human CD3(gamma) chain, human CD3(delta) chain,and human CD3(epsilon) chain) or a combination of two or more of theindividual human CD3 chains (e.g., a complex of human CD3(gamma) andhuman CD3(epsilon) or a complex of human CD3(delta) and humanCD3(epsilon)). In certain preferred embodiments, the Fab specificallybinds to a human CD3(epsilon) chain.

In certain other embodiments, a Fab of the present disclosurespecifically binds to TCR(alpha), TCR(beta), or a heterodimer formedfrom TCR(alpha) and TCR(beta). In certain embodiments, a Fabspecifically binds to one or more of human TCR(alpha), human TCR(beta),or a heterodimer formed from human TCR(alpha) and human TCR(beta).

In certain embodiments, a Fab of the present disclosure binds to acomplex formed from one or more CD3 chains with one or more TCR chains,such as a complex formed from a CD3(gamma) chain, a CD3(delta) chain, aCD3(epsilon) chain, a TCR(alpha) chain, or a TCR(beta) chain, or anycombination thereof. In other embodiments, a Fab of the presentdisclosure binds to a complex formed from one CD3(gamma) chain, oneCD3(delta) chain, two CD3(epsilon) chains, one TCR(alpha) chain, and oneTCR(beta) chain. In further embodiments, a Fab of the present disclosurebinds to a complex formed from one or more human CD3 chains with one ormore human TCR chains, such as a complex formed from a human CD3(gamma)chain, a human CD3(delta) chain, a human CD3(epsilon), a humanTCR(alpha) chain, or a human TCR(beta) chain, or any combinationthereof. In certain embodiments, a Fab of the present disclosure bindsto a complex formed from one human CD3(gamma) chain, one humanCD3(delta) chain, two human CD3(epsilon) chains, one human TCR(alpha)chain, and one human TCR(beta) chain.

Fabs of this disclosure can be generated as described herein or by avariety of methods known in the art (see, e.g., U.S. Pat. Nos.6,291,161; 6,291,158). Sources of Fabs include monoclonal antibodynucleic acid sequences from various species (which can be formatted asantibodies, Fvs, scFvs or Fabs, such as in a phage library), includinghuman, camelid (from camels, dromedaries, or llamas; Hamers-Casterman etal. (1993) Nature, 363:446 and Nguyen et al. (1998) J. Mol. Biol.,275:413), shark (Roux et al. (1998) Proc. Nat'l. Acad. Sci. (USA)95:11804), fish (Nguyen et al. (2002) Immunogenetics, 54:39), rodent,avian, or ovine.

An anti-human CD3 antibody with cross reactivity to monkey CD3 isparticularly desirable, such as the SP34 mouse monoclonal antibody,which binds specifically to human CD3 in denatured form (western blot ordot blot) and in native form (on T cells) (Pressano, S. The EMBO J.4:337-344, 1985; Alarcon, B. EMBO J. 10:903-912, 1991). SP34 mousemonoclonal antibody also binds to CD3ε singly transfected COS cells aswell as CD3ε/γ or CD3ε/δ double transfectants (Salmeron A. et al., J.Immunol. 147:3047-52, 1991). SP34 antibody also cross reacts non-humanprimates (Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad M L. etal., Cytometry 71A:925-33, 2007). In addition, SP34 activates T cellwhen cross-linked (Yang et al., J. Immunol. 137:1097-1100, 1986).Cross-reactivity to monkey CD3 is important as this allows toxicitystudies to be carried out in non-human primates using the clinicalcandidate directly, rather than in chimpanzee or using a surrogatemolecule. Thus, toxicity studies using such cross-reactive anti-CD3 Fabin an MSFP of the present disclosure provide more relevant safetyassessments.

Other illustrative anti-CD3 antibodies include the Cris-7 monoclonalantibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II., SpringerVerlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al.(1990) J. Exp. Med. 172:1691), OKT3 (Ortho multicenter Transplant StudyGroup (1985) N. Engl. J. Med. 313:337) and derivatives thereof such asOKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab(Carpenter et al. (2002) Blood 99:2712), and 145-2C11 monoclonalantibody (Hirsch et al. (1988) J. Immunol. 140: 3766). Further CD3binding molecules contemplated for use herein include UCHT-1 (Beverley,P C and Callard, R. E. (1981) Eur. J. Immunol. 11: 329-334) and CD3binding molecules described in WO2004/106380; WO2010/037838;WO2008/119567; WO2007/042261; WO2010/0150918.

An exemplary anti-TCR antibody is H57 monoclonal antibody (Lavasani etal. (2007) Scandinavian Journal of Immunology 65:39-47).

In certain embodiments, the Fab binds to other cell surface targets,including but not limited to, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa,FcγRIIIa, NKG2D, CD25, CD28, CD137, CTLA-4, FAS, FGFR1, FGFR2, FGFR3,FGFR4, GITR, LTβR, TLR, TRAIL receptor 1, TRAIL receptor 2, EGFR,Her2/neu, and ErbB3.

Antigen binding fragment sequences (e.g., heavy and light chain variableregion sequences) for Fab fragments may be available in public databasesor using traditional strategies for hybridoma development using a CD3chain, TCR component, or other Fab binding target as an immunogen inconvenient systems (e.g., mice, HuMAb mouse(R), TC mouse(TM),KM-mouse(R), llamas, chicken, rats, hamsters, rabbits, etc.) can be usedto develop Fabs for use herein. As would be understood by the skilledperson, Fab fragments may be generated using various technologies knownin the art, including antibody display technologies such as phage,yeast, ribosome and mRNA display technologies; B cell culture technologysuch as SLAM technology; or using high throughput gene sequencingtechnologies on B cells or plasma B cells isolated from an immunizedanimal subject or immunized human subject.

Illustrative Fabs for use in the MSFPs of the present disclosure includethe VH, Fd, HC, VL, and LC amino acid sequences, and the polynucleotidesencoding them, as set forth in SEQ ID NOs: 29-76, including CDRsthereof, such as those set forth in SEQ ID NOs: 23-28.

Fusion Moieties

The fusion moieties of the MSFP as described herein not only provideadditional binding specificities and/or functional attributes to theMSFP (e.g., increased serum half-life, activation of ADCC or otherimmune activation cascades), but also create steric hindrance tosignificantly reduce the binding of the Fab to its target antigen,except where/when intended (e.g. in the presence of tumor cells),particularly binding of the Fab to a target on a cell surface,especially Fab target antigens that are close to the cell membrane (suchas CD3) and therefore have restricted accessibility. This is in directcontrast to other Fab fusion proteins, such as TRIBODIES™ (i.e.,Fab-scFv fusion proteins), which fuse additional binding domains at theC terminus of the Fab fragment (see e.g., Journal of Immunology, 2000,165: 7050-7057).

Further, within a single MSFP, the first and second fusion moieties arenot intended to dimerize. This distinguishes the MSFP of the presentdisclosure from other known fusion proteins such as those described inWO2008/024188 and WO2009/149185. The constructs described in the '188and '185 publications differ by having the general format ofVH1-VH2-CH1-hinge-CH2-CH3 and VL1-VL2-CL. In this construct, the VH1 andVL1 dimerizes to form an additional antigen combining site. This differsfrom the format of the MSFP described herein, for example as shown inFIG. 7 Panel B. In the MSFP of the present disclosure, the first andsecond fusion moieties do not associate to form a single antigencombining site. A further distinguishing characteristic of the MSFP, asdescribed elsewhere herein, is that the fusion moieties reduce thebinding affinity of the Fab to its target when the MSFP is not clusteredon a target such as a cell surface target. On the contrary, the proteinsdescribed in WO2008/024188 and WO2009/149185 do not exhibit reducedbinding affinities for the antibody binding target.

The fusion moieties of the MSFP of the present disclosure may comprise abinding domain, one or more functional domains, or a combinationthereof. As depicted in FIG. 1, in one embodiment, the MSFP of thepresent disclosure comprise two fusion moieties, shown as Fusion moietyA and Fusion moiety B in FIG. 1. The fusion moieties may also bereferred to herein as a first fusion moiety and a second fusion moiety,or similar language to distinguish one from the other. Illustrativefusion moieties comprise the amino acid sequences set forth in SEQ IDNOs: 78, 88, and 94, encoded by the polynucleotide set forth in SEQ IDNOs: 77, 87, and 93; or a CDR, VH or VL thereof (see e.g. SEQ ID Nos:139-150).

In certain embodiments, the first and second fusion moieties areidentical. In further embodiments, both fusion moieties compriseidentical binding domains but may differ in other respects by having orlacking functional domains. In another embodiment, the first fusionmoiety comprises a first binding domain that binds a first targetantigen and the second fusion moiety comprises a second binding domainthat binds a second target antigen.

In certain embodiments, the first and second fusion moieties eachcomprise a binding domain and these may be referred to as the first andsecond binding domains, respectively. In certain embodiments, the firstbinding domain binds to the same target as the second binding domain. Inthis regard, the first binding domain may, in certain embodiments, bindto the same target as the second binding domain in the sense that thetargets share at least one similarity, for example without limitation,the targets comprise both protein, polynucleotide, or lipid, the targetsare the same protein though may be different splice forms; or in certainembodiments, the targets have the same amino acid sequence though mayhave different modifications, such as glycosylation. In certainembodiments, the first binding domain binds to the same target as thesecond binding domain but on different epitopes of the same target. Incertain embodiments, the first and second binding domains bind todifferent targets.

In another embodiment, the first fusion moiety comprises a first bindingdomain and the second fusion moiety comprises a second binding domainwherein the first and second binding domains bind the same targetmolecule but are of different formats (e.g., the first binding domain isan scFv that binds to a cell surface receptor and the second bindingdomain is a ligand for the receptor; or similarly, the first bindingdomain is an scFv that binds to a ligand, the second binding domain isan extracellular domain of the receptor for the ligand).

Binding Domains

As noted above, in certain embodiments, the first and/or second fusionmoieties comprise a binding domain. A “binding domain” or “bindingregion” according to the present disclosure may be, for example, anyprotein, polypeptide, oligopeptide, or peptide that possesses theability to specifically recognize and bind to a biological molecule(e.g., a cell surface receptor or tumor protein, or a componentthereof). A binding domain includes any naturally occurring, synthetic,semi-synthetic, or recombinantly produced binding partner for abiological molecule of interest. For example, and as further describedherein, a binding domain may be antibody light chain and heavy chainvariable region regions, or the light and heavy chain variable regionregions can be joined together in a single chain and in eitherorientation (e.g., VL-VH or VH-VL). A variety of assays are known foridentifying binding domains of the present disclosure that specificallybind with a particular target, including Western blot, ELISA, flowcytometry, or surface plasmon resonance analysis (e.g., using BIACORE™analysis).

Illustrative binding domains are described further herein below. Incertain embodiments, the target molecule may be a cell surface expressedprotein, such as a receptor or a tumor antigen. In another embodiment,the target molecule bound by a binding domain useful herein is a solubleantigen such as a cytokine, albumin, or other serum protein.Illustrative binding domains include immunoglobulin antigen-bindingdomains such as scFv, scTCR, extracellular domains of receptors, ligandsfor cell surface molecules/receptors, or receptor binding domainsthereof, and tumor binding proteins. In certain embodiments, the antigenbinding domains can be an scFv, a VH, a VL, a domain antibody variant(dAb), a camelid antibody (VHH), a fibronectin 3 domain variant, anankyrin repeat variant and other antigen-specific binding domain derivedfrom other protein scaffolds.

Thus, in certain embodiments, a binding domain comprises anantibody-derived binding domain but can be a non-antibody derivedbinding domain. An antibody-derived binding domain can be a fragment ofan antibody or a genetically engineered product of one or more fragmentsof the antibody, which fragment is involved in binding with the antigen.Examples include, without limitation, a complementarity determiningregion (CDR), a variable region (Fv), a heavy chain variable region(VH), a light chain variable region (VL), a heavy chain, a light chain,a single chain variable region (scFv), a Fab, a single domain camelantibody (camelid VHH), and single domain antibodies (dAb).

As would be understood by the skilled person and as described elsewhereherein, a complete antibody comprises two heavy chains and two lightchains. Each heavy chain consists of a variable region and a first,second, and third constant region, while each light chain consists of avariable region and a constant region. Mammalian heavy chains areclassified as α, δ, ε, γ, and μ, and mammalian light chains areclassified as λ, or κ. Immunoglobulins comprising the α, δ, ε, γ, and μheavy chains are classified as Immunoglobulin (Ig)A, IgD, IgE, IgG, andIgM. The complete antibody forms a “Y” shape. The stem of the Y consistsof the second and third constant regions (and for IgE and IgM, thefourth constant region) of two heavy chains bound together and disulfidebonds (inter-chain) are formed in the hinge. Heavy chains γ, α and δhave a constant region composed of three tandem (in a line) Ig domains,and a hinge region for added flexibility; heavy chains μ and ε have aconstant region composed of four immunoglobulin domains. The second andthird constant regions are referred to as “CH2 domain” and “CH3 domain”,respectively. Each arm of the Y includes the variable region and firstconstant region of a single heavy chain bound to the variable andconstant regions of a single light chain. The variable regions of thelight and heavy chains are responsible for antigen binding.

“Complementarity determining region” or “CDR” with regard to an antibodyrefers to a highly variable loop in the variable region of the heavychain or the light chain of an antibody. CDRs can interact with theantigen conformation and largely determine binding to the antigen(although some framework regions are known to be involved in binding).The heavy chain variable region and the light chain variable region eachcontain 3 CDRs. The CDRs can be defined or identified by conventionalmethods, such as by sequence according to Kabat et al (Wu, TT and Kabat,E. A., J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat E. A.,PNAS, 84: 2440-2443 (1987); Kabat, E. A. et al, Sequences of proteins ofimmunological interest, Published by DIANE Publishing, 1992), or bystructure according to Chothia et al (Choithia, C. and Lesk, A. M., JMol. Biol., 196(4): 901-917 (1987), Choithia, C. et al, Nature, 342:877-883 (1989)).

“Heavy chain variable region” or “VH” with regard to an antibody refersto the fragment of the heavy chain that contains three CDRs interposedbetween flanking stretches known as framework regions, which are morehighly conserved than the CDRs and form a scaffold to support the CDRs.

“Light chain variable region” or “VL” with regard to an antibody refersto the fragment of the light chain that contains three CDRs interposedbetween framework regions.

“Fv” with regard to an antibody refers to the smallest fragment of theantibody to bear the complete antigen binding site. An Fv fragmentconsists of the variable region of a single light chain bound to thevariable region of a single heavy chain.

“Single-chain Fv antibody” or “scFv” with regard to an antibody refersto an engineered antibody consisting of a light chain variable regionand a heavy chain variable region connected to one another directly orvia a peptide linker sequence.

“Single domain camel antibody” or “camelid VHH” as used herein refers tothe smallest known antigen-binding unit of a heavy chain antibody(Koch-Nolte, et al, FASEB J., 21: 3490-3498 (2007)). A “heavy chainantibody” or a “camelid antibody” refers to an antibody that containstwo VH domains and no light chains (Riechmann L. et al, J. Immunol.Methods 231:25-38 (1999); WO94/04678; WO94/25591; U.S. Pat. No.6,005,079).

“Single domain antibody” or “dAb” refers to an antibody fragment thatconsists of the variable region of an antibody heavy chain (VH domain)or the variable region of an antibody light chain (VL domain) (Holt, L.,et al, Trends in Biotechnology, 21(11): 484-490).

The term “disulfide bond” as used herein refers to the binding of aheavy chain fragment and a light chain fragment through one or moredisulfide bonds. The one or more disulfide bonds can be formed betweenthe two fragments by linking the thiol groups in the two fragments. Incertain embodiments, the one or more disulfide bonds can be formedbetween one or more cysteine residues in the heavy chain fragment andthe light chain fragment, respectively.

A “variable region linking sequence” is an amino acid sequence thatconnects a heavy chain variable region to a light chain variable regionand provides a spacer function compatible with interaction of the twosub-binding domains so that the resulting polypeptide retains a specificbinding affinity to the same target molecule as an antibody thatcomprises the same light and heavy chain variable regions. In certainembodiments, a hinge useful for linking a binding domain to animmunoglobulin CH2 or CH3 region polypeptide may be used as a variableregion linking sequence (see further discussion of hinges elsewhereherein).

In certain embodiments, a binding domain comprises a non-antibodycomponent. The non-antibody component which binds to an antigen can beany suitable protein domain or components that can recognize and bind toa target antigen of interest, such as for example, protein domains thatinvolve in protein-protein interactions, in protein-lipid interactions,in protein-polynucleotide interactions, in protein-sugar interactions,or in ligand binding. Examples of suitable non-antibody componentinclude, without limitation, Fibronectin 3 domain (Fn3), an ankyrinrepeat, and an Adnectin.

An alternative source of binding domains of this disclosure includessequences that encode random peptide libraries or sequences that encodean engineered diversity of amino acids in loop regions of alternativenon-antibody scaffolds, such as fibrinogen domains (see, e.g., Weisel etal. (1985) Science 230:1388), Kunitz domains (see, e.g., U.S. Pat. No.6,423,498), lipocalin domains (see, e.g., WO 2006/095164), V-likedomains (see, e.g., US Patent Application Publication No. 2007/0065431),C-type lectin domains (Zelensky and Gready (2005) FEBS J. 272:6179), orFcab™ (see, e.g., PCT Patent Application Publication Nos. WO2007/098934; WO 2006/072620), or the like.

In certain embodiments, a binding domain comprises an Fn3 domain.“Fibronectin 3 domain” or “Fn3” as used herein refers to an autonomousfolding unit in fibronectin which is involved in binding to biologicalmolecules (Calaycay, J. et al, J. Biol. Chem., 260(22): 12136-41 (1985);Koide, A. et al, J. Mol. Biol., 284(4): 1141-1151 (1998); Bloom, L. etal, Drug Discovery Today, 14(19-20): 949-955 (2009)). The Fn3 domain canbe found in a variety of proteins and different repeats of Fn3 domainare found to contain binding sites for biological molecules such as DNAand proteins.

In certain embodiments, a binding domain comprises adnectin. “Adnectin”as used herein refers to a genetically engineered protein that is basedon an Fn3 domain (Koide, A. et al, Methods Mol. Biol., 352: 95-109(2007)). The Fn3 domain in Adnectin contains three loops that mimics thethree CDRs of the variable region of an antibody, and can be geneticallytailored for specific binding to different target molecules.

In certain embodiments, a binding domain comprises an ankyrin repeat.“Ankyrin repeat” as used herein refers to a protein component containingrepeats of a 33-amino acid residue found in erythrocyte ankyrin (Davis,L. H. et al, J. Biol. Chem., 266(17): 11163-11169 (1991)). Ankyrinrepeat is known as one of the most common protein-protein interactionstructure that occurs in a large number of proteins with differentfunctions.

As depicted in the Figures, scFv are particularly illustrative bindingdomains. The scFv as used as a binding domain of a fusion moiety maybind to any of a variety of target molecules, including but not limitedto FcγRI, FcγRIIa FcγRIIb FcγRIIIa FcγRIIIb, CD28, CD137, CTLA-4, FAS,fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4,glucocorticoid-induced TNFR-related (GITR) protein, lymphotoxin-betareceptor (LTβR), toll-like receptors (TLR), tumor necrosisfactor-related apoptosis-inducing ligand-receptor 1 (TRAIL receptor 1)and TRAIL receptor 2, carcino-embryonic antigen (CEA), prostate-specificmembrane antigen (PSMA) protein, prostate stem cell antigen (PSCA)protein, B cell maturation antigen (BCMA; also known as human tumornecrosis factor receptor superfamily member 17 (TNFRSF17) and CD269),tumor-associated protein carbonic anhydrase IX (CAIX), hepatocyte growthfactor receptor (HGFR), epidermal growth factor receptor 1 (EGFR1),human epidermal growth factor receptor 2 (Her2/neu; Erb2), ErbB3,epithelial cell adhesion molecule (EpCAM), Folate receptor, Ephrinreceptors, CD19, CD20, CD30, CD33, CD40, CD37, CD38, and CD138.

In certain embodiments, the binding domain binds to a tumor antigen.Illustrative tumor antigen target molecules include, without limitation,p53, cMet, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10,MAGE-A12, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B,NA88-A, NY-ESO-1, BRCA1, BRCA2, MART-1, MC1R, Gp100, PSA, PSM,Tyrosinase, Wilms' tumor antigen (VVT1), TRP-1, TRP-2, ART-4, CAMEL,CEA, Cyp-B, Her2/neu, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RU1,RU2, SART-1, SART-3, WT1, AFP, β-catenin/m, Caspase-8/m, CEA, CDK-4/m,ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3,Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDCl27/m,TPI/mbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARα, and TEL/AML1. These andother tumor proteins are known to the skilled artisan.

Other binding domains useful in the fusion moieties of the MSFP of thepresent disclosure include ligands which bind to cell surface receptors.Illustrative ligands include, but are not limited to, ligands for cellsurface receptors such as CD28, CD137, CTLA-4, FAS, fibroblast growthfactor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid-inducedTNFR-related (GITR) protein, lymphotoxin-beta receptor (LTβR), toll-likereceptors (TLR), tumor necrosis factor-related apoptosis-inducingligand-receptor 1 (TRAIL receptor 1) and TRAIL receptor 2,carcino-embryonic antigen (CEA), prostate-specific membrane antigen(PSMA) protein, prostate stem cell antigen (PSMA) protein, B cellmaturation antigen (BCMA; also known as human tumor necrosis factorreceptor superfamily member 17 (TNFRSF17) and CD269), tumor-associatedprotein carbonic anhydrase IX (CAIX), hepatocyte growth factor receptor(HGFR), epidermal growth factor receptor 1 (EGFR1), human epidermalgrowth factor receptor 2 (Her2/neu; Erb2), ErbB3, epithelial celladhesion molecule (EpCAM), Folate receptor, Ephrin receptor, CD19, CD20,CD30, CD33, CD40, CD37, CD38, and CD138.

In one embodiment, a binding domain binds to serum albumin. Binding tohuman serum albumin offers the potential to extend the half life(t_(1/2)) of a protein drug to several days or even longer. It is knownin the prior art that serum albumin binding or fusion to serum albumincan significantly extend the half life of a therapeutic molecule. Incertain embodiments, the binding domain which binds to human serumalbumin can cross react with the non-human primate orthologue,permitting estimation of the half life of the drug in preclinical animaltoxicological studies that may be more relevant to treatment in humans.

In certain embodiments, the binding domain specifically binds to anantigen target that is associated with a disease condition. The diseasecondition may include a physiological condition, a pathologicalcondition and a cosmetic condition. Examples of illustrative conditionsinclude, without limitation, cancer, inflammatory disorders, allografttransplantation, type I diabetes, type II diabetes, and multiplesclerosis.

In certain embodiments, the antigen target is negatively associated withthe condition. In certain embodiments, the binding of the antigen targetby an MSFP comprising a binding domain can inactivate or antagonize thebiological function of the antigen target, and thereby improve thecondition. In certain embodiments, the binding of the antigen target byan MSFP comprising a binding domain will activate or antagonize thebiological function of the antigen target, and thereby improve thecondition. As such the MSFPs described herein may be agonist orantagonist molecules with respect to the fusion moiety target antigens.

Illustrative binding domains are provided in SEQ ID NOs: 78, 88, 94(amino acid) and SEQ ID NOs: 77, 87, 93 (polynucleotide) and includeCDRs, VH, and VL thereof (see e.g. SEQ ID Nos: 139-150).

Thus, the binding domains as described herein can specifically bind toany suitable antigen targets. As noted, examples of suitable antigentargets include, without limitation, TNF receptor (Shen H. M. et al,FASEB J. 20(10):1589-98 (2006)), cMet (Bottaro, D. P. et al, Science,251 (4995): 802-804 (1991)), CD3 (Chetty R. et al, J Pathol., 173(4):303-7 (1994)), CD40 (Chatzigeorgiou A. et al, Biofactors., 35(6): 474-83(2009)), DR3 (Meylan F. et al, Immunity., 29(1):79-89 (2008)), FcγR(Torkildsen O. et al, Acta Neurol Scand Suppl. 183:61-3 (2006)), NKG2D(Obeidy P. et al, Int J Biochem Cell Biol., 41(12):2364-7 (2009)), andany derivative thereof.

In certain embodiments, the binding domains bind specifically to asingle target antigen. In another embodiment, the binding domains arecross-reactive with more than one antigen target. “Cross-reactivity” asused herein refers to that a binding domain can specifically bind tomore than one antigen target. In certain embodiments, the first bindingdomain and/or the second binding domain can have cross-reactivity tocompletely different antigen targets, such as for example, hepatitis Ccore protein and host-derived GOR protein. In certain embodiments, thefirst binding domain and/or the second binding domain can havecross-reactivity to an antigen target from a different species, such asfor example, an antigen derived from a protein from human, mouse ornonhuman primates.

Junction Between Fusion Moiety A, B and Fab

In certain embodiments, fusion moiety A and/or fusion moiety B arelinked directly to the N-terminus of the VH or VL of the Fab (i.e., withno additional amino acids added between). In other embodiments, fusionmoiety A and/or fusion moiety B are linked to the N-terminus of the VHor VL of the Fab using a linker (with additional amino acids asdescribed below). In some embodiments, it may be necessary to deleteseveral amino acids (e.g., from 1-3 amino acids or from 1-10 aminoacids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) from theC-terminus of a given fusion moiety A and/or B, depending on the Fabtarget and the surrounding space of the Fab target on the cell surface(accessibility of the Fab target on the cell surface).

In other embodiments, it may be necessary to delete several amino acids(e.g., from 1-3 amino acids or from 1-10 amino acids; e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 amino acids) from the N-terminus of the heavyand/or light chain of the Fab. In yet further embodiments, it may benecessary to delete several amino acids (e.g., from 1-3 amino acids orfrom 1-10 amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 aminoacids) from the C-terminus of the fusion moiety and at the same time, todelete several amino acids (e.g., from 1-3 amino acids or from 1-10amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) fromthe N-terminus of the Fab chain. The length and the sequence of thejunction between fusion moiety A, fusion moiety B and the Fab fragmentcan be the same or different.

The junction between the fusion moieties and the Fab fragment may makeuse of a combination of deletions and linkers as needed. As would beunderstood by the skilled artisan, the junction between the Fab and thefusion moieties can be adjusted accordingly and tested for desiredfunctionality (e.g., binding affinity) using methods known in the artand described herein.

Linkers

The MSFP of the present disclosure may also comprise a linker situatedbetween the VH and the VL of the Fab fragment and the fusion moieties Aand B. (See e.g., FIG. 1). And illustrative linker comprises thesequence Gly-Arg-Ala.

In one embodiment, the linker between a fusion moiety and the Fab VH orVL is 1-10 amino acids long. In other embodiments, the linker between afusion moiety and the Fab VH or VL is 1-20 or 20 amino acids long. Inthis regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In furtherembodiments, the linker may be 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30amino acids long.

In certain embodiments, linkers suitable for use in the MSFP describedherein are flexible linkers. Suitable linkers can be readily selectedand can be of any of a suitable of different lengths, such as from 1amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 aminoacids, from 3 amino acids to 12 amino acids, including 4 amino acids to10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 aminoacids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6,or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)(SEQ ID NO: 125) and (GGGS)_(n) (SEQ ID NO: 126), where n is an integerof at least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers known in the art. Glycine and glycine-serinepolymers are relatively unstructured, and therefore may be able to serveas a neutral tether between components. Glycine accesses significantlymore phi-psi space than even alanine, and is much less restricted thanresidues with longer side chains (see Scheraga, Rev. Computational Chem.11173-142 (1992)). Exemplary flexible linkers include, but are notlimited to Gly-Gly-Ser-Gly (SEQ ID NO: 127), Gly-Gly-Ser-Gly-Gly (SEQ IDNO: 128), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 129), Gly-Ser-Gly-Gly-Gly (SEQID NO: 130), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 131), Gly-Ser-Ser-Ser-Gly(SEQ ID NO: 132), and the like. The ordinarily skilled artisan willrecognize that design of an MSFP can include linkers that are all orpartially flexible, such that the linker can include a flexible linkeras well as one or more portions that confer less flexible structure toprovide for a desired MSFP structure.

In certain embodiments, the linker between the Fab and the fusionmoieties is a stable linker (not cleavable by protease, especiallyMMPs). In certain embodiments, the linker is a peptide linker. Incertain embodiments, the MSFP comprises a stable peptide linker, and theN-terminal of the peptide linker is covalently linked to the C-terminalof the fusion moiety, and the C terminal of the peptide linker iscovalently linked to the N-terminal of the antigen-binding domain.

In one embodiment, the linker is a cleavable linker. In particular, thelinker between the Fab VH or VL and a fusion moiety comprises a proteasesubstrate cleavage sequence, for example, an MMP substrate cleavagesequence. A well known peptide sequence of PLGLAG (SEQ ID NO: 133) in asubstrate can be cleaved by most MMPs.

Substrate sequences that can be cleaved by MMPs have been extensivelystudied. For example, the sequence of PLGLAG (SEQ ID NO: 133) can becleaved by most MMPs. A protease substrate cleavage sequence refers to apeptide sequence that can be cleaved by protease treatment. An MMPsubstrate sequence refers to a peptide sequence that can be cleaved byincubation with a MMP. PLGLAG (SEQ ID NO: 133) is a commonly used MMPsubstrate cleavage sequence (see e.g., Jiang, PNAS (2004) 101:17867-72;Olson, PNAS (2010) 107:4311-6). In another embodiment, the proteasecleavage site is recognized by MMP-2, MMP-9 or a combination thereof. Inyet another embodiment, the protease site comprises the sequenceselected from the group consisting of GPLGMLSQ (SEQ ID NO: 134) andGPLGLWAQ (SEQ ID NO: 135). A stable linker or a protease non cleavablelinker refers to a linker peptide sequence that does not belong to theknown protease substrate sequences and thus does not lead to significantcleavage product formation upon incubation with a protease.

In some embodiments, the cleavage substrate (or cleavage sequence) ofthe linker may include an amino acid sequence that can serve as asubstrate for a protease, usually an extracellular protease. In otherembodiments, the cleavage sequence comprises a cysteine-cysteine paircapable of forming a disulfide bond, which can be cleaved by action of areducing agent. In other embodiments the cleavage sequence comprises asubstrate capable of being cleaved upon photolysis.

The cleavage substrate is positioned in the linker such that when thecleavage substrate is cleaved by a cleaving agent (e.g., a cleavagesubstrate of a linker is cleaved by the protease and/or thecysteine-cysteine disulfide bond is disrupted via reduction by exposureto a reducing agent) or by light-induced photolysis, in the presence ofa target, resulting in cleavage products having various functionalproperties as described herein.

The cleavage substrate of a linker may be selected based on a proteasethat is co-localized in the diseased tissue, or on the surface of thecell that expresses the target antigen of interest of a binding domainof a fusion moiety. A variety of different conditions are known in whicha target of interest is co-localized with a protease, where thesubstrate of the protease is known in the art. In the example of cancer,the target tissue can be a cancerous tissue, particularly canceroustissue of a solid tumor. There are reports in the literature ofincreased levels of proteases having known substrates in a number ofcancers, e.g., solid tumors. See, e.g., La Rocca et al, (2004) BritishJ. of Cancer 90(7): 1414-1421. Non-limiting examples of disease include:all types of cancers (breast, lung, colorectal, prostate, head and neck,pancreatic, etc.), rheumatoid arthritis, Crohn's disease, melanomas,SLE, cardiovascular damage, ischemia, etc. Furthermore, anti-angiogenictargets, such as VEGF, are known. As such, where the binding domain of afusion moiety of the MSFP of the present disclosure is selected suchthat it is capable of binding a tumor antigen, a suitable cleavagesubstrate sequence for the linker will be one which comprises a peptidesubstrate that is cleavable by a protease that is present at thecancerous treatment site, particularly that is present at elevatedlevels at the cancer treatment site as compared to non-canceroustissues. In one exemplary embodiment, the binding domain of an MSFP canbind, e.g., Her2 and the cleavage substrate sequence can be a matrixmetalloprotease (MMP) substrate, and thus is cleavable by an MMP. Inother embodiments, the binding domain of a fusion moiety in the MSFP canbind a target of interest and the cleavage substrate present in thelinker can be, for example, legumain, plasmin, TMPRSS-3/4, MMP-9,MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase,uPA, or PSA. In other embodiments, the cleave substrate is cleaved byother disease-specific proteases, in diseases other than cancer such asmultiple sclerosis or rheumatoid arthritis.

The unmodified or uncleaved linker can allow for tethering the Fabfragment to the fusion moiety. When the linker is cleaved, multiplecleavage products with various functions may result as described furtherherein for example, in the Figures.

The linkers of the MSFP (e.g., the linker between the VH of the Fab anda first fusion moiety and the linker between the VL of the Fab and asecond fusion moiety) can comprise the same cleavage substrate or maycomprise different cleavage substrates, e.g., the first linker maycomprise a first cleavage substrate and the second linker may comprise asecond cleavage substrate. The first and second cleavage substrates canbe different substrates for the same enzyme (for example exhibitingdifferent binding affinities to the enzyme), or different substrates fordifferent enzymes, or the first cleavage substrate can be an enzymesubstrate and the second cleavage substrate can be a photolysissubstrate, or the first cleavage substrate can be an enzyme substrateand the second cleavage substrate can be a substrate for reduction, andthe like.

For specific cleavage by an enzyme, contact between the enzyme and thecleavage substrate is made. When the MSFP comprising a Fab coupled to afirst and a second fusion moiety by first and second linkers havingcleavage substrates in the presence sufficient enzyme activity, thecleavage substrate can be cleaved. Sufficient enzyme activity can referto the ability of the enzyme to make contact with the linker having thecleavage substrate and effect cleavage. It can readily be envisionedthat an enzyme may be in the vicinity of the MSFP but unable to cleavebecause of other cellular factors or protein modification of the enzyme.

Exemplary substrates can include but are not limited to substratescleavable by one or more of the following enzymes or proteases: ADAM10;Caspase 8, Cathepsin S, MMP 8, ADAM12, Caspase 9, FAP, MMP 9, ADAM17,Caspase 10, Granzyme B, MMP-13, ADAMTS, Caspase 11, Guanidinobenzoatase(GB), MMP 14, ADAMTS5. Caspase 12, Hepsin, MT-SP1, BACE, Caspase 13,Human Neutrophil Elastase Neprilysin (HNE), Caspases, Caspase 14,Legumain, NS3/4A, Caspase 1, Cathepsins, Matriptase 2, Plasmin, Caspase2, Cathepsin A, Meprin, PSA, Caspase 3, Cathepsin B, MMP 1, PSMA,Caspase 4, Cathepsin D, MMP 2, TACE, Caspase 5, Cathepsin E, MMP 3,TMPRSS ¾, Caspase 6, Cathepsin K, MMP 7, uPA, Caspase 7, MT1-MMP.

In another embodiment, the cleavage substrate can involve a disulfidebond of a cysteine pair, which is thus cleavable by a reducing agentsuch as, for example, but not limited to a cellular reducing agent suchas glutathione (GSH), thioredoxins, NADPH, flavins, ascorbate, and thelike, which can be present in large amounts in tissue of or surroundinga solid tumor.

Other appropriate protease cleavage sites for use in the cleavablelinkers herein are known in the art or may be identified using methodssuch as those described by Turk et al., 2001 Nature Biotechnology 19,661-667.

In certain embodiments, the linker can be a peptide linker, a thiolresidue-containing peptide linker, such as a cysteine residue, a polymerlinker or a chemical linker. In certain embodiments, the MSFP comprisesa linker where one end of the linker is covalently linked to theC-terminal of the fusion moiety, and the other end of the linker iscovalently linked to the N-terminal of the VH or VL of the Fab fragment.

Junctional Amino Acids

In certain embodiments, there may be one or a few amino acid residuesbetween two domains of a MSFP, such as between a binding domain and alinker polypeptide, such as amino acid residues resulting from constructdesign of the fusion protein (e.g., amino acid residues resulting fromthe use of a restriction enzyme site during the construction of anucleic acid molecule encoding a single chain polypeptide). As describedherein, such amino acid residues may be referred to “junction aminoacids” or “junction amino acid residues”, or “peptide spacers”.

In certain illustrative embodiments, a peptide spacer is between 1 to 5amino acids, between 5 to 10 amino acids, between 5 to 25 amino acids,between 5 to 50 amino acids, between 10 to 25 amino acids, between 10 to50 amino acids, between 10 to 100 amino acids, or any intervening rangeof amino acids. In other illustrative embodiments, a peptide spacercomprises about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more aminoacids in length.

Such junctional amino acids link any of the domains of the MSFP Incertain embodiments, the junctional amino acid(s) is a hinge or a partof a hinge as defined herein. In certain embodiments, a variable regionlinking sequence useful for connecting a heavy chain variable region toa light chain variable region may be used as a peptide spacer.

In one illustrative embodiment, peptide spacer sequences contain, forexample, Gly, Asn and Ser residues. Other near neutral amino acids, suchas Thr and Ala, may also be included in the spacer sequence.

Other amino acid sequences which may be usefully employed as spacersinclude those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphyet al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. Nos.4,935,233 and 4,751,180.

Other illustrative spacers may include, for example,Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO:136)(Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) andLys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp(SEQ ID NO:137) (Bird et al., 1988, Science 242:423-426).

In some embodiments, spacer sequences are not required when the firstand second polypeptides have non-essential N-terminal amino acid regionsthat can be used to separate the functional domains and prevent stericinterference. Two coding sequences or domains of the MSFP of the presentdisclosure can be fused directly without any junctional amino acids orby using a flexible polylinker composed, for example, of the pentamerGly-Gly-Gly-Gly-Ser (SEQ ID NO:138) repeated 1 to 3 times. Such a spacerhas been used in constructing single chain antibodies (scFv) by beinginserted between VH and VL (Bird et al., 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5979-5883).

A peptide spacer, in certain embodiments, is designed to enable thecorrect interaction between two beta-sheets forming the variable regionof the single chain antibody.

Any suitable linkers can be used to make an indirect link, such aswithout limitation, peptide linker, polymer linker, and chemical linker.In certain embodiments, the covalent link is an indirect link through apeptide linker.

It should be noted that, within a single MSFP, the first and secondfusion moieties do not dimerize. This distinguishes the MSFP of thepresent disclosure from other known fusion proteins such as thosedescribed in WO2008/024188 and WO2009/149185. These constructs differ byhaving the general format of VH1-VH2-CH1-hinge-CH2-CH3 and VL1-VL2-CL.In this construct, the VH1 and VL1 dimerizes to form an additionalantigen combining site. In the MSFP of the present disclosure, the firstand second fusion moieties do not associate to form a single antigencombining site. A further distinguishing characteristic of the MSFP, asdescribed elsewhere herein, is that the fusion moieties reduce thebinding affinity of the Fab to its target when the MSFP is notclustered. On the contrary, the proteins described in WO2008/024188 andWO2009/149185 do not exhibit reduced binding affinities for the antibodybinding target.

Illustrative MSFP of the present disclosure comprise any one of theamino acid sequences selected from SEQ ID NOs: 84, 90, 96, and 100; andany one of the amino acid sequences selected from SEQ ID NOs: 32, 60,64, 68, and 72. Additional illustrative MSFP of the present disclosurecomprise any one of the amino acid sequences selected from SEQ ID NOs:86, 92, 98, and 102; and any one of the amino acid sequences selectedfrom SEQ ID NOs: 30, 36, 40, 44, 48, and 52.

Function of the Multi-Specific Fab Fusion Proteins

The MSFP of the present disclosure functions to enhance drug stability,specificity, selectivity, potency, and safety and the convenience ofdrug administration. In certain embodiments, the Fab, when expressedwithout the fusion moieties fused to its N-terminus, is able to bind toits target antigen in soluble recombinant form (usually theextracellular domain of a receptor protein, e.g., a T cell receptorcomponent such as CD3) as well as on the cell surface. In certainembodiments, the Fab antigen-binding fragment, when expressed withfusion moieties fused to its N-termini of both heavy and light chainsand in a monomeric form (non-aggregated or not multimeric form), has nobinding or has reduced binding or similar level of binding as Fab aloneto its specific antigen presented on cell surface at pharmacologicalconcentrations of the drug (concentration of the polypeptide in treatedpatients) in the absence of target antigen binding by a fusion moietybinding domain. Lack of binding or greatly reduced binding to cellsurface antigen in the absence of the target antigen binding by a fusionmoiety binding domain may be explained by the dramatically reducedaffinity resulting from the designed steric hindrance around the antigenbinding site.

Lack of binding or greatly reduced binding to cell surface antigen inthe absence of the target antigen binding by a fusion moiety bindingdomain may be viewed as desirable for MSFP as a human therapeutic. It isimportant to note that lack of binding or significantly reduced bindingof MSFP alone (in the absence of tumor target cells) to, e.g., T cellcan, 1) dramatically improve the undesirable systematic T cellactivation, therefore to dramatically improve the drug safety profile;2) dramatically improves the feasibility of subcutaneous route of drugadministration; and 3) dramatically increase the drug tolerability ofhigh drug concentration in blood circulation.

It is important to note that T cell binding by antibodies such as OKT3or UCHT-1 via conformational epitopes may transduce partial signaling,leading either to unwanted T cell activation (causing cytokine storm) orT cell anergy (resulting in T cells unable to kill tumor cells). Mu-1F3,hu-1F3 and its variants binding to a linear epitope of CD3 isconceivably less likely to induce T cell signaling in the absence ofcross linking of the CD3. This property may be advantageous for reducingsystemic side effects that occur when using OKT3 and UCHT-1 likeantibodies.

It is also important to note that once a fusion moiety in a MSFP iscleaved by protease, it functions such that the steric hindrance aroundthe Fab antigen-binding site is removed so that it can then bind to its(Fab) target with high affinity, particularly target antigen expressedon the cell surface. Therefore, following cleavage at the cleavagesubstrate sequence in a linker (thereby releasing a fusion moiety) theMSFP is converted into a more potent cross linker between tumor and Tcells.

Furthermore, it is important to note that once a fusion moiety bindingdomain binds to its target antigen, the MSFP molecules become highlyconcentrated on tumor cell surface to create high avidity based bindingtoward the Fab target (e.g. CD3) on T cells. Therefore, only in thepresence of the fusion moiety binding is the Fab antigen-bindingfragment able to bind its target thus for MSFP to function as across-linker between tumor and T cells.

The properties of the MSFP of the present disclosure allow forrelatively high dose of the MSFP in circulation without unwantedside-effects (e.g., the MSFP does not bind to the Fab fragment targetantigen (e.g., CD3) when in circulation. This also allows for reduceddosing frequency and promotes tissue penetration by diffusion driven byconcentration gradient.

The properties of the MSFP of the present disclosure also allow thepotential for the subcutaneous administration which can enhance accessto the target. Further, although in certain embodiments the MSFP arepermissive for cross linking without protease treatment, in certainparticular embodiments, the binding activity and the tumor killingpotency increase dramatically after protease treatment.

In one embodiment, the antigen binding domain (Fv) formed by VH and VLis stabilized by the CH1 and CL heterdimerizing domain, and is furtherstabilized by the disulfide bond, or other stabilizing interaction(e.g., knobs/hole interaction), between CH1 and CL.

In one embodiment, the Fab in the MSFP is sterically hindered by thefusion moieties at its N-termini such that binding to the Fab targetantigen (especially when cell surface target antigens are concerned) isreduced in a statistically significant manner (i.e., relative to anappropriate control as will be known to those skilled in the art; e.g.,as compared to the same Fab in a format without fusion moieties at itsN-termini (both VH and VL)). In a further embodiment, the Fab in theMSFP is sterically hindered by the fusion moieties at its N-termini sothe binding to the desirable antigen (especially when cell surfacetarget antigens are concerned) is reduced by at least 2 fold, 3 fold, 4fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold,13 fold, 14 fold, 15 fold, 20 fold, 30 fold, or 100 fold, or 1000 fold,or 10,000 fold as compared to the same Fab in a format without fusionmoieties at its N-termini (both VH and VL).

In certain embodiments, the affinity of the Fab antigen-binding domainin the fusion protein format for the Fab cell surface target antigen isbelow 500 nM. In further embodiments, the affinity of the Fab antigenbinding domain in the fusion protein format (i.e., the MSFP)demonstrates no significant detectible binding as measured using FACS orother binding measurement method (e.g., cell binding ELISA) atconcentration ranges of the therapeutics used in humans. In oneembodiment, less than 1% of a population of Fab target cells (e.g., CD3+cells) will be bound by the Fab fusion protein at a therapeuticconcentration (this is in the absence of cells expressing a fusionmoiety binding domain target antigen). In one embodiment, less than 5%population of the Fab target cells will be bound by the MSFP at atherapeutic concentration. In yet another embodiment, less than 10%population of the Fab target cells will be bound by the MSFP at atherapeutic concentration.

The elevated level of proteases, especially MMPs, present in tumortissues will generate cleavage products at the MMP substrate cleavagesite of a linker. Because the cleavage of the protease substratesequence of a linker results in the relief of the steric hindrance atthe Fab antigen-binding region, the binding to the Fab cell surfacetarget will be fully restored or at least partially restored. Therestored binding can be demonstrated using techniques of FACS,cell-based ELISA) or other cell binding techniques known to the skilledperson.

The term “dramatically reduced affinity” refers to at least 30%reduction in the binding of the Fab antigen-binding domain, as comparedto the binding when the N-termini of the VH and VL of the Fab are freeof fusion moieties. The percentage of reduction can be, for examplewithout limitation, 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or about 99% or greater. Methods for detectingbinding are known to the skilled person and can be performed using FACS,cell binding ELISA or cell binding using radioisotope labeledantibodies.

The illustrative Fab for use in the MSFP of the present disclosure asdescribed herein is an anti-CD3 Fab. In this regard, the MSFP functionssuch that, when the fusion moiety binding domain binds to a tumor cellantigen, the Fab is able bind to CD3 of passing T cells, therebyredirecting the T cells and activating them to kill the tumor cell. Inanother embodiment, the MSFP (also referred to herein as Fabe where theFab fragment binds to an immune effect or molecule, such as CD3) canexhibit an avidity effect when clustered on tumor cell surface via tumorantigen binding by fusion moiety binding domain(s). As such, theapparent binding to immune cells by the sterically hindered Fab canincrease due to avidity. As such, the Fabe/MSFP becomes capable ofbridging immune and tumor cells thereby mediating anti-tumor activity.

In certain embodiments, separate binding of the Fab to its targetantigen or a fusion moiety binding agent binding to its target antigendoes not lead to target activation. However, when simultaneously bound,the Fab antigen target and the fusion moiety binding domain antigentarget can generate signal transduction. For example, an MSFP can bindto a Fab antigen target which is, e.g., CD3, and a fusion moiety bindingdomain antigen target which is a tumor surface antigen. When the MSFP isseparately bound to either CD3 or tumor surface antigen, the T cellswill not be activated, however, when the CD3 and tumor surface antigenare simultaneously bound to the MSFP and when multiple copies of thebound complexes are anchored and clustered on tumor cell surface, the Tcells are activated in the vicinity of cancer cells bearing the tumorsurface antigen, and therefore significantly enhance the tumor killingefficiency of T cells locally and avoid the side effects due to cytokinestorm.

In certain embodiments, the combination of the Fab antigen target andthe fusion moiety binding domain antigen target can be CD3 and tumorsurface antigen, which combination can enhance tumor killing effects byT cells. In certain embodiments, the combination of the Fab antigentarget and the fusion moiety antigen target can be FcγR and tumorsurface antigen, which combination can induce FcγR-expressing immunecells to kill tumor cells. In certain embodiments, the combination ofthe Fab antigen target and the fusion moiety antigen target can be NKG2Dand a tumor cell surface antigen, which combination can induce naturalkiller (NK) cell to kill tumor cells.

In certain embodiments, the first and second fusion moieties comprisebinding domains that bind a first and second target antigen. In thisway, the MSFP binds three different target antigens (i.e., the Fabtarget plus two different fusion moiety targets). Thus, in certainembodiments, the Fab antigen target is selected from the groupconsisting of CD3, TCR, FcγR and NKG2D, and the first and second bindingdomains of the first and second fusion moieties are two differentantigens preferentially expressed on cancer cells. Such an MSFP havingthree different target antigens may enhance the targeting specificityfor tumor cells and prevent killing of normal cells that may express oneof the fusion moiety binding domain target antigens or that may expresslow levels of both fusion moiety binding domain target antigens.

Thus, in an illustrative embodiment, the MSFP of the present disclosurecomprises a Fab antigen-binding fragment that binds to the TCR or acomponent thereof, such as a CD3 polypeptide. As noted above, the MSFPof the present disclosure does not bind to the Fab target antigen exceptwhen fusion moiety binding domains engage their target antigen orfollowing a linker cleavage event.

Thus, in certain embodiments, an MSFP of the present disclosure does notor minimally activates T cells in the absence of fusion moiety bindingdomain target antigen engagement. An MSFP “does not or minimally ornominally activates T cells” if the MSFP does not cause a statisticallysignificant increase in the percentage of activated T cells as comparedto activation of T cells in the presence of cells expressing fusionmoiety binding domain target antigens (e.g., an appropriate tumorcell/cell line), as measured in at least one in vitro or in vivo assay.Such assays are known in the art and include, without limitation,proliferation assays, CTL chromium release assays (see e.g., Lavie etal., (2000) International Immunology 12(4):479-486), ELISPOT assays,intracellular cytokine staining assays, and others as described, forexample, in Current Protocols in Immunology, John Wiley & Sons, NewYork, N.Y. (2009). In certain embodiments, T cell activation is measuredusing and in vitro primed T cell activation assay. See also assays asdescribed in the examples herein.

In a related aspect, therefore, the present disclosure provides a methodfor detecting T cell activation induced by the MSFP that comprises a Fabthat specifically bindings to a TCR complex or a component thereof,comprising: (a) providing antigen or mitogen-primed T cells, (b)treating the primed T cells of step (a) with the MSFP that comprises aFab that specifically binds to a TCR complex or a component thereof, and(c) detecting activation of the primed T cells that have been treated instep (b).

The term “mitogen” as used herein refers to a chemical substance thatinduces mitosis in lymphocytes of different specificities or clonalorigins. Exemplary mitogens that may be used to prime T cells includephytohaemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide(LPS), pokeweed mitogen (PWM), and phorbol myristate acetate (PMA).Antigen-loaded beads or PBMC can also be used to prime T cells.

In certain embodiments of methods for detecting T cell activationprovided herein, the MSFP comprising a Fab that specifically binds to aTCR complex or a component thereof comprises one or more fusion moietiescomprising one or more binding domains that bind to tumor antigens.

T cell activation may be detected by measuring the expression ofactivation markers known in the art, such as CD25, CD40 ligand, andCD69. Activated T cells may also be detected by cell proliferationassays, such as CFSE labeling and thymidine uptake assays (Adams (1969)Exp. Cell Res. 56:55). T cell effector function (e.g., cell killing) canbe measured, for example, by chromium release assays or FACS basedassays using fluorescent dyes (e.g. TP3),In a related aspect, T cellactivation and cytolytic activity can be measured by lytic synapseformation between T cell and tumor cell. Effector molecules such asGranzymes and perforin can be detected in the cytolytic synapse.

In another related aspect, T cell activation may be measured by cytokinerelease. A method for detecting cytokine release induced by an MSFP thatcomprises a Fab that specifically binds to a TCR complex or a componentthereof, may comprise: (a) providing primed T cells, (b) treating theprimed T cells of step (a) with the MSFP that comprises a Fab thatspecifically binds to a TCR complex or a component thereof, and (c)detecting release of a cytokine from the primed T cells that have beentreated in step (b). In particular embodiments, experiments are carriedout in the presence or absence of appropriate cancer cells or cell linesexpressing target tumor antigens bound by binding domains present in thefirst and/or second fusion moieties of the MSFP.

In certain embodiments of methods for detecting cytokine releaseprovided herein, the MSFP that comprises a Fab that specifically bindsto a TCR complex or a component thereof is an MSFP that furthercomprises fusion moieties that comprise binding domains that bind to atumor target antigen.

In further preferred embodiments, the MSFP of the present disclosure donot induce a cytokine storm or do not induce a cytokine releasesufficient to induce toxic side-effects. An MSFP “does not induce acytokine storm” (also referred to as “inducing an undetectable, nominal,or minimal cytokine release” or “does not induce or induces a minimallydetectable cytokine release”) if, in the absence of secondary targetcells (e.g., tumor cells expressing antigens bound by binding domain ofthe fusion moieties) or appropriate linker cleavage agents (such asproteases), it does not cause a statistically significant increase inthe amount of at least one cytokine including IFNγ; In certainembodiments at least two cytokines including IFNγ and TNFα or IL-6 andTNFα; in one embodiment three cytokines including IL-6, IFNγ, and TNFα;in another embodiment four cytokines including IL-2, IL-6, IFNγ, andTNFα; and in yet a further embodiment at least five cytokines includingIL-2, IL-6, IL-10, IFNγ, and TNFα; released from treated cells in theabsence of secondary target cells (e.g., an appropriate cancer cellline) or appropriate linker cleavage agents, as compared to from treatedcells in the presence of appropriate secondary target cells or linkercleavage agents, in at least one in vitro or in vivo assay known in theart or provided herein. Clinically, cytokine-release syndrome ischaracterized by fever, chills, rash, nausea, and sometimes dyspnea andtachycardia, which is in parallel with maximal release of certaincytokines, such as IFNγ, as well as IL-2, IL-6, and TNFα. Cytokines thatmay be tested for release in an in vitro assay or in vivo include G-CSF,GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IP-10, KC, MCP1,IFNγ, and TNFα; and in another embodiment include IL-2, IL-6, IL-10,IFNγ, and TNFα.

In further embodiments, an MSFP of the present disclosure causes anincrease in calcium flux in cells, such as T cells. An MSFP causes an“increase in calcium” if, when used to activate T cells in the presenceof an appropriate secondary target cell (e.g., cancer cell) or linkercleavage agents, it causes a statistically significant, rapid increasein calcium flux of the treated cells (preferably within 300 seconds,more preferably within 200 seconds, and most preferably within 100seconds of treatment) as compared to cells treated in the absence of anappropriate secondary target cell or linker cleavage agents, as measuredin an in vitro assay known in the art or provided herein.

In further embodiments, an MSFP of the present disclosure inducesphosphorylation of a molecule in the TCR signal transduction pathway.The “TCR signal transduction pathway” refers to the signal transductionpathway initiated via the binding of a peptide:MHC ligand to the TCR andits co-receptor (CD4 or CD8). A “molecule in the TCR signal transductionpathway” refers to a molecule that is directly involved in the TCRsignal transduction pathway, such as a molecule whose phosphorylationstate (e.g., whether the molecule is phosphorylated or not), whosebinding affinity to another molecule, or whose enzymatic activity, hasbeen changed in response to the signal from the binding of a peptide:MHCligand to the TCR and its co-receptor. Exemplary molecules in the TCRsignal transduction pathway include the TCR complex or its components(e.g., CD3ζ chains), ZAP-70, Fyn, Lck, phospholipase c-γ, protein kinaseC, transcription factor NFκB, phosphatase calcineurin, transcriptionfactor NFAT, guanine nucleotide exchange factor (GEF), Ras, MAP kinasekinase kinase (MAPKKK), MAP kinase kinase (MAPKK), MAP kinase (ERK1/2),and Fos.

An MSFP of this disclosure “induces phosphorylation of a molecule in theTCR signal transduction pathway” if it causes a statisticallysignificant increase in phosphorylation of a molecule in the TCR signaltransduction pathway (e.g., CD3 chains, ZAP-70, and ERK1/2) only in thepresence of appropriate secondary target antigens, or cells expressingsuch antigen (e.g., cancer cells expressing tumor antigens bound byfusion moiety binding domains) or linker cleavage agents, in an in vitroor in vivo assay or receptor signaling assays known in the art. Resultsfrom most receptor signaling assays known in the art are determinedusing immunohistochemical methods, such as western blots or fluorescencemicroscopy.

Similarly, the MSFP of the present disclosure induce killing ofsecondary target cell, such as tumor cells expressing fusion moietybinding domain target antigens, by T cells. Such cell killing can bemeasured using a variety of assays known in the art, including chromiumrelease assays.

The specificity and function of an MSFP of the present disclosure may betested by contacting the MSFP with appropriate test sample and, incertain embodiments, treating the MSFP with an appropriate proteasewhich is thought to be specific for the cleavage recognition site in thelinker and assaying for cleavage products. Proteases may be isolated,for example from cancer cells or they may be prepared recombinantly, forexample following the procedures in Darket et al. (J. Biol. Chem.254:2307-2312 (1988)). The cleavage products may be identified forexample based on size, antigenicity or activity. The toxicity of theMSFP may be investigated by subjecting the MSFP and cleavage productsthereof to in vitro cytotoxicity, proliferation, binding, or otherappropriate assays known to the skilled person. Toxicity of the cleavageproducts may be determined using a ribosomal inactivation assay (Westbyet al., Bioconjugate Chem. 3:377-382 (1992)). The effect of the cleavageproducts on protein synthesis may be measured in standardized assays ofin vitro translation utilizing partially defined cell free systemscomposed for example of a reticulocyte lysate preparation as a source ofribosomes and various essential cofactors, such as mRNA template andamino acids. Use of radiolabeled amino acids in the mixture allowsquantitation of incorporation of free amino acid precursors intotrichloroacetic acid precipitable proteins. Rabbit reticulocyte lysatesmay be conveniently used (O'Hare, FEBS Lett. 273:200-204 (1990)).

The ability of the MSFP of the invention to destroy cancer cells and/oractivate T cells may be readily tested in vitro using cancer cell lines,T cell lines or isolated PBMC or T cells. The effects of the MSFP of thepresent disclosure may be determined, for example, by demonstrating byselective lysis of cancer cells. In addition, the protease specificitycan be tested by comparing the inhibition of cellular proliferationusing an MSFP of the present disclosure alone or in the presence ofprotease-specific inhibitors. Such protease inhibitors may includeMMP-2/MMP-9 inhibitors GM1489, GM6001 and GI-I to GI-IV.

Toxicity may also be measured based on cell viability, for example theviability of normal and cancerous cell cultures exposed to the MSFP maybe compared. Cell viability may be assessed by known techniques, such astrypan blue exclusion assays. Toxicity may also be measured based oncell lysis, for example the lysis of normal and cancerous cell culturesexposed to the MSFP may be compared. Cell lysis may be assessed by knowntechniques, such as Chromium (Cr) release assays or dead cell indicatordyes (propidium Iodide, TOPRO®3 iodide, i.e. a carbocyanine monomernucleic acid stain).

Polypeptides

The present disclosure provides MSFP polypeptides, and fragmentsthereof. Illustrative polypeptides, and the polynucleotides encodingthem, are provided in SEQ ID NOs: 23-102 and 109-150. He The terms“polypeptide” “protein” and “peptide” and “glycoprotein” are usedinterchangeably and mean a polymer of amino acids not limited to anyparticular length. The term does not exclude modifications such asmyristoylation, sulfation, glycosylation, phosphorylation and additionor deletion of signal sequences. The terms “polypeptide” or “protein”means one or more chains of amino acids, wherein each chain comprisesamino acids covalently linked by peptide bonds, and wherein saidpolypeptide or protein can comprise a plurality of chains non-covalentlyand/or covalently linked together by peptide bonds, having the sequenceof native proteins, that is, proteins produced by naturally-occurringand specifically non-recombinant cells, or genetically-engineered orrecombinant cells, and comprise molecules having the amino acid sequenceof the native protein, or molecules having deletions from, additions to,and/or substitutions of one or more amino acids of the native sequence.The terms “polypeptide” and “protein” specifically encompass the MSFPand dimers thereof of the present disclosure, or sequences that havedeletions from, additions to, and/or substitutions of one or more aminoacid of an MSFP as disclosed herein. Thus, a “polypeptide” or a“protein” can comprise one (termed “a monomer”) or a plurality (termed“a multimer”) of amino acid chains.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldtypically be found in nature, (2) is essentially free of other proteinsfrom the same source, e.g., from the same species, (3) is expressed by acell from a different species, (4) has been separated from at leastabout 50 percent of polynucleotides, lipids, carbohydrates, or othermaterials with which it is associated in nature, (5) is not associated(by covalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Such an isolated protein can be encoded by genomic DNA, cDNA,mRNA or other RNA, of may be of synthetic origin, or any combinationthereof. In certain embodiments, the isolated protein is substantiallyfree from proteins or polypeptides or other contaminants that are foundin its natural environment that would interfere with its use(therapeutic, diagnostic, prophylactic, research or otherwise).

The term “polypeptide fragment” refers to a polypeptide, which can bemonomeric or multimeric, that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion or substitutionof a naturally-occurring or recombinantly-produced polypeptide. Incertain embodiments, a polypeptide fragment can comprise an amino acidchain at least 5 to about 500 amino acids long. It will be appreciatedthat in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200,250, 300, 350, 400, or 450 amino acids long. Particularly usefulpolypeptide fragments include functional domains, includingantigen-binding domains or fragments of antibodies. In the case of ananti-CD3, or other antibody, useful fragments include, but are notlimited to: a CDR region, especially a CDR3 region of the heavy or lightchain; a variable region of a heavy or light chain; a portion of anantibody chain or just its variable region including two CDRs; and thelike.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be fused in-frame or conjugated to a linker or other sequencefor ease of synthesis, purification or identification of the polypeptide(e.g., poly-His), or to enhance binding of the polypeptide to a solidsupport.

Amino acid sequence modification(s) of the MSFPs described herein arecontemplated. For example, it may be desirable to improve the bindingaffinity and/or other biological properties of the MSFP. For example,amino acid sequence variants of an MSF, or binding domain or Fab thereofmay be prepared by introducing appropriate nucleotide changes into apolynucleotide that encodes the MSFP, or a domain thereof, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the MSFP. Any combination of deletion,insertion, and substitution may be made to arrive at the final MSFP,provided that the final construct possesses the desired characteristics,such as specific binding to a target antigen of interest by a bindingdomain or Fab. The amino acid changes also may alter post-translationalprocesses of the MSFP, such as changing the number or position ofglycosylation sites. Any of the variations and modifications describedabove for polypeptides of the present invention may be included inantibodies of the present invention.

The present disclosure provides variants of the MSFP disclosed herein.In certain embodiments, such variant MSFP comprise variant bindingdomains or Fab fragments thereof, or antigen-binding fragments, or CDRsthereof, bind to a target of interest at least about 50%, at least about70%, and in certain embodiments, at least about 90% as well as a givenreference or wild-type sequence, including any such sequencesspecifically set forth herein. In further embodiments, such variantsbind to a target antigen with greater affinity the reference orwild-type sequence set forth herein, for example, that bindquantitatively at least about 105%, 106%, 107%, 108%, 109%, or 110% aswell as a reference sequence specifically set forth herein.

In certain embodiments, the present disclosure provides variants of theMSFPs disclosed herein where such variants comprise Fabs that have beenmodified with regard to the disulfide bond between the VH and VL. Aswould be recognized by the skilled person, in certain embodiments theFab fragment used in the MSFP of the present invention may not comprisea disulfide bond. In this regard, the heavy and light chains may beengineered in such a way so as to stably interact without the need fordisulfide bond. For example, in certain embodiments, the heavy or lightchain can be engineered to remove a cysteine residue and wherein theheavy and light chains still stably interact and function as a Fab. Inone embodiment, mutations are made to facilitate stable interactionbetween the heavy and light chains. For example, a “knobs into holes”engineering strategy can be used to facilitate dimerization between theheavy and light chains of a Fab (see e.g., 1996 Protein Engineering,9:617-621). Thus, also contemplated for use herein are variant Fabfragments designed for a particular purpose, for example, removal of adisulfide bond addition of tax for purification, etc.

In particular embodiments, a subject MSFP may have: an amino acidsequence that is at least 80% identical, at least 95% identical, atleast 90%, at least 95% or at least 98% or 99% identical, to the MSFPdescribed herein.

Determination of the three-dimensional structures of representativepolypeptides may be made through routine methodologies such thatsubstitution, addition, deletion or insertion of one or more amino acidswith selected natural or non-natural amino acids can be virtuallymodeled for purposes of determining whether a so derived structuralvariant retains the space-filling properties of presently disclosedspecies. See, for instance, Donate et al., 1994 Prot. Sci. 3:2378;Bradley et al., Science 309: 1868-1871 (2005); Schueler-Furman et al.,Science 310:638 (2005); Dietz et al., Proc. Nat. Acad. Sci. USA 103:1244(2006); Dodson et al., Nature 450:176 (2007); Qian et al., Nature450:259 (2007); Raman et al. Science 327:1014-1018 (2010). Someadditional non-limiting examples of computer algorithms that may be usedfor these and related embodiments, include VMD which is a molecularvisualization program for displaying, animating, and analyzing largebiomolecular systems using 3-D graphics and built-in scripting (see thewebsite for the Theoretical and Computational Biophysics Group,University of Illinois at Urbana-Champagne, atks.uiuc.edu/Research/vmd/. Many other computer programs are known in theart and available to the skilled person and which allow for determiningatomic dimensions from space-filling models (van der Waals radii) ofenergy-minimized conformations; GRID, which seeks to determine regionsof high affinity for different chemical groups, thereby enhancingbinding, Monte Carlo searches, which calculate mathematical alignment,and CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER(Weiner et al (1981) J. Comput Chem. 106: 765), which assess force fieldcalculations, and analysis (see also, Eisenfield et al. (1991) Am. J.Physiol. 261:C376-386; Lybrand (1991) J. Pharm. Belg. 46:49-54;Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins7:99-111; Pedersen (1985) Environ. Health Perspect 61:185-190; and Kiniet al. (1991) J. Biomol. Struct. Dyn. 9:475-488). A variety ofappropriate computational computer programs are also commerciallyavailable, such as from Schrödinger (Munich, Germany).

Polynucleotides Encoding the Multi-Specific Fab FusionProteins/Vectors/Host Cells/and Methods of Making Multi-Specific FabFusion Proteins

The present disclosure further provides in certain embodiments anisolated nucleic acid encoding the polypeptide MSFP as described herein.Illustrative polynucleotides, and the polypeptides encoded thereby, anand fragments thereof, are provided in SEQ ID NOs: 23-102 and 109-150.Nucleic acids include DNA and RNA. These and related embodiments mayinclude polynucleotides encoding the MSFP as described herein. The term“isolated polynucleotide” as used herein shall mean a polynucleotide ofgenomic, cDNA, or synthetic origin or some combination thereof, which byvirtue of its origin the isolated polynucleotide (1) is not associatedwith all or a portion of a polynucleotide in which the isolatedpolynucleotide is found in nature, (2) is linked to a polynucleotide towhich it is not linked in nature, or (3) does not occur in nature aspart of a larger sequence.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, atranscription control sequence “operably linked” to a protein codingsequence is ligated thereto so that expression of the protein codingsequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “control sequence” as used herein refers to polynucleotidesequences that can affect expression, processing or intracellularlocalization of coding sequences to which they are ligated or operablylinked. The nature of such control sequences may depend upon the hostorganism. In particular embodiments, transcription control sequences forprokaryotes may include a promoter, ribosomal binding site, andtranscription termination sequence. In other particular embodiments,transcription control sequences for eukaryotes may include promoterscomprising one or a plurality of recognition sites for transcriptionfactors, transcription enhancer sequences, transcription terminationsequences and polyadenylation sequences. In certain embodiments,“control sequences” can include leader sequences and/or fusion partnersequences.

The term “polynucleotide” as referred to herein means single-stranded ordouble-stranded nucleic acid polymers. In certain embodiments, thenucleotides comprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine,ribose modifications such as arabinoside and 2′,3′-dideoxyribose andinternucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term“polynucleotide” specifically includes single and double stranded formsof DNA.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups and the like. Theterm “oligonucleotide linkages” includes oligonucleotide linkages suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al., 1986, Nucl.Acids Res., 14:9081; Stec et al., 1984, J. Am. Chem. Soc., 106:6077;Stein et al., 1988, Nucl. Acids Res., 16:3209; Zon et al., 1991,Anti-Cancer Drug Design, 6:539; Zon et al., 1991, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, pp. 87-108 (F. Eckstein, Ed.), OxfordUniversity Press, Oxford England; Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman, 1990, Chemical Reviews, 90:543, the disclosures ofwhich are hereby incorporated by reference for any purpose. Anoligonucleotide can include a detectable label to enable detection ofthe oligonucleotide or hybridization thereof.

In other related embodiments, polynucleotide variants may havesubstantial identity to a polynucleotide sequence encoding an MSFP, ordomain thereof as described herein. For example, a polynucleotide may bea polynucleotide comprising at least 70% sequence identity, preferablyat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher,sequence identity compared to a reference polynucleotide sequence suchas a sequence encoding an MSFP or domain thereof described herein, usingthe methods described herein, (e.g., BLAST analysis using standardparameters, as described below). One skilled in this art will recognizethat these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the binding affinity of a binding domain, or binding affinity of aFab, or function of the MSFP encoded by the variant polynucleotide isnot substantially diminished relative to the unmodified referenceprotein encoded by a polynucleotide sequence specifically set forthherein.

In certain other related embodiments, polynucleotide fragments maycomprise or consist essentially of various lengths of contiguousstretches of sequence identical to or complementary to a sequenceencoding an MSFP or domain thereof as described herein. For example,polynucleotides are provided that comprise or consist essentially of atleast about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,140, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of asequences the encodes an MSFP or domain thereof, such as a bindingdomain or Fab antigen-binding fragment thereof, disclosed herein as wellas all intermediate lengths there between. It will be readily understoodthat “intermediate lengths”, in this context, means any length betweenthe quoted values, such as 50, 51, 52, 53, etc.; 100, 101, 102, 103,etc.; 150, 151, 152, 153, etc.; including all integers through 200-500;500-1,000, and the like. A polynucleotide sequence as described here maybe extended at one or both ends by additional nucleotides not found inthe native sequence. This additional sequence may consist of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotidesat either end of a polynucleotide encoding an MSFP or domain thereofdescribed herein or at both ends of a polynucleotide encoding an MSFP ordomain thereof described herein.

In another embodiment, polynucleotides are provided that are capable ofhybridizing under moderate to high stringency conditions to apolynucleotide sequence an MSFP or domain thereof, such as a bindingdomain or a Fab antigen-binding fragment thereof, provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide as provided herein with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60° C.-65° C. or 65° C.-70° C.

In certain embodiments, the polynucleotides described above, e.g.,polynucleotide variants, fragments and hybridizing sequences, encode anMSFP or domain thereof, such as a binding domain or a Fab, e.g., a Fabthat binds CD3 or a binding domain that binds a tumor antigen target. Inother embodiments, such polynucleotides encode MSFP that bind to CD3and/or a tumor antigen at least about 50%, at least about 70%, and incertain embodiments, at least about 90% as well as an MSFP sequencespecifically set forth herein. In further embodiments, suchpolynucleotides encode an MSFP or domain thereof, that, e.g., bind toCD3 and/or a target antigen with greater affinity than the MSFP, ordomain thereof, set forth herein, for example, that bind quantitativelyat least about 105%, 106%, 107%, 108%, 109%, or 110% as well as an MSFP,or domain thereof, sequence specifically set forth herein.

As described elsewhere herein, determination of the three-dimensionalstructures of representative polypeptides (e.g., variant MSFP asprovided herein, for instance, an MSFP having a binding domain and a Fabas provided herein) may be made through routine methodologies such thatsubstitution, addition, deletion or insertion of one or more amino acidswith selected natural or non-natural amino acids can be virtuallymodeled for purposes of determining whether a so derived structuralvariant retains the space-filling properties of presently disclosedspecies. A variety of computer programs are known to the skilled artisanfor determining appropriate amino acid substitutions (or appropriatepolynucleotides encoding the amino acid sequence) within, for example,an antibody or antigen-binding fragment thereof, such that, for example,affinity is maintained or better affinity is achieved.

The polynucleotides described herein, or fragments thereof, regardlessof the length of the coding sequence itself, may be combined with otherDNA sequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, illustrative polynucleotide segments with totallengths of about 10,000, about 5000, about 3000, about 2,000, about1,000, about 500, about 200, about 100, about 50 base pairs in length,and the like, (including all intermediate lengths) are contemplated tobe useful.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegAlign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., Unified Approach to Alignment and Phylogenes, pp. 626-645 (1990);Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., CABIOS 5:151-153 (1989); Myers, E. W.and Muller W., CABIOS 4:11-17 (1988); Robinson, E. D., Comb. Theor11:105 (1971); Santou, N. Nes, M., Mol. Biol. Evol. 4:406-425 (1987);Sneath, P. H. A. and Sokal, R. R., Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.(1973); Wilbur, W. J. and Lipman, D. J., Proc. Natl. Acad., Sci. USA80:726-730 (1983).

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman, Add.APL. Math 2:482 (1981), by the identity alignment algorithm of Needlemanand Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similaritymethods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444(1988), by computerized implementations of these algorithms (GAP,BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.),or by inspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977), and Altschul et al., J. Mol. Biol.215:403-410 (1990), respectively. BLAST and BLAST 2.0 can be used, forexample with the parameters described herein, to determine percentsequence identity among two or more the polynucleotides. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

In certain embodiments, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) of 20 percent or less, usually 5 to 15percent, or 10 to 12 percent, as compared to the reference sequences(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The percentage is calculated by determining thenumber of positions at which the identical nucleic acid bases occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thereference sequence (i.e., the window size) and multiplying the resultsby 100 to yield the percentage of sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode an MSFP as described herein. Some of thesepolynucleotides bear minimal sequence identity to the nucleotidesequence of the native or original polynucleotide sequence that encodeMSFP, for example an MSFP that binds to CD3 and or a tumor targetantigen. Nonetheless, polynucleotides that vary due to differences incodon usage are expressly contemplated by the present disclosure. Incertain embodiments, sequences that have been codon-optimized formammalian expression are specifically contemplated.

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, may be employed for thepreparation of variants and/or derivatives of the MSFP described herein.By this approach, specific modifications in a polypeptide sequence canbe made through mutagenesis of the underlying polynucleotides thatencode them. These techniques provides a straightforward approach toprepare and test sequence variants, for example, incorporating one ormore of the foregoing considerations, by introducing one or morenucleotide sequence changes into the polynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments, the inventors contemplate the mutagenesis of thepolynucleotide sequences that encode an MSFP disclosed herein, or adomain thereof, to alter one or more properties of the encodedpolypeptide, such as the binding affinity of a binding domain or the Fabantigen-binding fragment thereof, or the function of a particular Fcregion, or the affinity of the Fc region for a particular FcγR. Thetechniques of site-specific mutagenesis are well-known in the art, andare widely used to create variants of both polypeptides andpolynucleotides. For example, site-specific mutagenesis is often used toalter a specific portion of a DNA molecule. In such embodiments, aprimer comprising typically about 14 to about 25 nucleotides or so inlength is employed, with about 5 to about 10 residues on both sides ofthe junction of the sequence being altered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants,recursive sequence recombination, as described in U.S. Pat. No.5,837,458, may be employed. In this approach, iterative cycles ofrecombination and screening or selection are performed to “evolve”individual polynucleotide variants having, for example, increasedbinding affinity. Certain embodiments also provide constructs in theform of plasmids, vectors, transcription or expression cassettes whichcomprise at least one polynucleotide as described herein.

In certain embodiments, the isolated polynucleotide is inserted into avector. The term “vector” as used herein refers to a vehicle into whicha polynucleotide encoding a protein may be covalently inserted so as tobring about the expression of that protein and/or the cloning of thepolynucleotide. The isolated polynucleotide may be inserted into avector using any suitable methods known in the art, for example, withoutlimitation, the vector may be digested using appropriate restrictionenzymes and then may be ligated with the isolated polynucleotide havingmatching restriction ends.

Examples of suitable vectors include, without limitation, plasmids,phagemids, cosmids, artificial chromosomes such as yeast artificialchromosome (YAC), bacterial artificial chromosome (BAC), or P1—derivedartificial chromosome (PAC), bacteriophages such as lambda phage or M13phage, and animal viruses. Examples of categories of animal virusesuseful as vectors include, without limitation, retrovirus (includinglentivirus), adenovirus, adeno-associated virus, herpesvirus (e.g.,herpes simplex virus), poxvirus, baculovirus, papillomavirus, andpapovavirus (e.g., SV40).

For expression of the polypeptide, the vector may be introduced into ahost cell to allow expression of the polypeptide within the host cell.The expression vectors may contain a variety of elements for controllingexpression, including without limitation, promoter sequences,transcription initiation sequences, enhancer sequences, selectablemarkers, and signal sequences. These elements may be selected asappropriate by a person of ordinary skill in the art. For example, thepromoter sequences may be selected to promote the transcription of thepolynucleotide in the vector. Suitable promoter sequences include,without limitation, T7 promoter, T3 promoter, SP6 promoter, beta-actinpromoter, EF1a promoter, CMV promoter, and SV40 promoter. Enhancersequences may be selected to enhance the transcription of thepolynucleotide. Selectable markers may be selected to allow selection ofthe host cells inserted with the vector from those not, for example, theselectable markers may be genes that confer antibiotic resistance.Signal sequences may be selected to allow the expressed polypeptide tobe transported outside of the host cell.

A vector may also include materials to aid in its entry into the cell,including but not limited to a viral particle, a liposome, or a proteincoating.

For cloning of the polynucleotide, the vector may be introduced into ahost cell (an isolated host cell) to allow replication of the vectoritself and thereby amplify the copies of the polynucleotide containedtherein. The cloning vectors may contain sequence components generallyinclude, without limitation, an origin of replication, promotersequences, transcription initiation sequences, enhancer sequences, andselectable markers. These elements may be selected as appropriate by aperson of ordinary skill in the art. For example, the origin ofreplication may be selected to promote autonomous replication of thevector in the host cell.

In certain embodiments, the present disclosure provides isolated hostcells containing the vector provided herein. The host cells containingthe vector may be useful in expression or cloning of the polynucleotidecontained in the vector.

Suitable host cells can include, without limitation, prokaryotic cells,fungal cells, yeast cells, or higher eukaryotic cells such as mammaliancells.

Suitable prokaryotic cells for this purpose include, without limitation,eubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescens, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such asP. aeruginosa, and Streptomyces.

The expression of antibodies and antigen-binding fragments inprokaryotic cells such as E. coli is well established in the art. For areview, see for example Pluckthun, A. Bio/Technology 9: 545-551 (1991).Expression in eukaryotic cells in culture is also available to thoseskilled in the art as an option for production of antibodies orantigen-binding fragments thereof, see recent reviews, for example Ref,M. E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J. J. et al.(1995) Curr. Opinion Biotech 6: 553-560.

Suitable fungal cells for this purpose include, without limitation,filamentous fungi and yeast. Illustrative examples of fungal cellsinclude, Saccharomyces cerevisiae, common baker's yeast,Schizosaccharomyces pombe, Kluyveromyces hosts such as, e.g., K. lactis,K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickerhamii(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906),K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichiapastoris (EP 183,070); Candida; Trichoderma reesei (EP 244,234);Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis;and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

Higher eukaryotic cells, in particular, those derived from multicellularorganisms can be used for expression of glycosylated polypeptideprovided herein. Suitable higher eukaryotic cells include, withoutlimitation, invertebrate cells and insect cells, and vertebrate cells.Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruit fly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the K−1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells. Plant cell cultures of cotton, corn, potato, soybean,petunia, tomato, and tobacco can also be utilized as hosts. Examples ofvertebrate cells include, mammalian host cell lines such as monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRK-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

The vector can be introduced to the host cell using any suitable methodsknown in the art, including, without limitation, DEAE-dextran mediateddelivery, calcium phosphate precipitate method, cationic lipids mediateddelivery, liposome mediated transfection, electroporation,microprojectile bombardment, receptor-mediated gene delivery, deliverymediated by polylysine, histone, chitosan, and peptides. Standardmethods for transfection and transformation of cells for expression of avector of interest are well known in the art.

In certain embodiments, the host cells comprise a first vector encodinga first polypeptide and a second vector encoding a second polypeptide.In certain embodiments, the first vector and the second vector may bethe same or not the same. In certain embodiments, the first polypeptideand the second polypeptide may be the same or not the same.

In certain embodiments, the first vector and the second vector may ormay not be introduced simultaneously. In certain embodiments, the firstvector and the second vector may be introduced together into the hostcell. In certain embodiments, the first vector may be introduced firstinto the host cell, and then the second vector may be introduced. Incertain embodiments, the first vector may be introduced into the hostcell which is then established into a stable cell line expressing thefirst polypeptide, and then the second vector may be introduced into thestable cell line.

In certain embodiments, the host cells comprise a vector encoding for afirst polypeptide and a second polypeptide. In certain embodiments, thefirst polypeptide and the second polypeptide may be the same or not thesame.

In certain embodiments, the present disclosure provides methods ofexpressing the polypeptide provided herein, comprising culturing thehost cell containing the vector under conditions in which the insertedpolynucleotide in the vector is expressed.

Suitable conditions for expression of the polynucleotide may include,without limitation, suitable medium, suitable density of host cells inthe culture medium, presence of necessary nutrients, presence ofsupplemental factors, suitable temperatures and humidity, and absence ofmicroorganism contaminants. A person with ordinary skill in the art canselect the suitable conditions as appropriate for the purpose of theexpression.

In certain embodiments, the polypeptide expressed in the host cell canform a dimer and thus produce an MSFP dimer or polypeptide complexprovided herein. In certain embodiments, the polypeptide expressed inthe host cell can form a polypeptide complex which is a homodimer. Incertain embodiments, where the host cells express a first polynucleotideand a second polynucleotide, the first polynucleotide and the secondpolynucleotide can form a polypeptide complex which is a heterodimer.

In certain embodiments, the polypeptide complex may be formed inside thehost cell. For example, the dimer may be formed inside the host cellwith the aid of relevant enzymes and/or cofactors. In certainembodiments, the polypeptide complex may be secreted out of the cell. Incertain embodiments, the first polypeptide and the second polypeptidemay be secreted out of the host cell and form a dimer outside of thehost cell.

In certain embodiments, the first polypeptide and the second polypeptidemay be separately expressed and allowed to dimerize under suitableconditions. For example, the first polypeptide and the secondpolypeptide may be combined in a suitable buffer and allow the firstprotein monomer and the second protein monomer to dimerize throughappropriate interactions such as hydrophobic interactions. For anotherexample, the first polypeptide and the second polypeptide may becombined in a suitable buffer containing an enzyme and/or a cofactorwhich can promote the dimerization of the first polypeptide and thesecond polypeptide. For another example, the first polypeptide and thesecond polypeptide may be combined in a suitable vehicle and allow themto react with each other in the presence of a suitable reagent and/orcatalyst.

The expressed polypeptide and/or the polypeptide complex can becollected using any suitable methods. The polypeptide and/or thepolypeptide complex can be expressed intracellularly, in the periplasmicspace or be secreted outside of the cell into the medium. If thepolypeptide and/or the polypeptide complex is expressed intracellularly,the host cells containing the polypeptide and/or the polypeptide complexmay be lysed and polypeptide and/or the polypeptide complex may beisolated from the lysate by removing the unwanted debris bycentrifugation or ultrafiltration. If the polypeptide and/or thepolypeptide complex is secreted into periplasmic space of E. coli, thecell paste may be thawed in the presence of agents such as sodiumacetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) overabout 30 min, and cell debris can be removed by centrifugation (Carteret al., BioTechnology 10:163-167 (1992)). If the polypeptide and/or thepolypeptide complex is secreted into the medium, the supernatant of thecell culture may be collected and concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. A protease inhibitor and/or aantibiotics may be included in the collection and concentration steps toinhibit protein degradation and/or growth of contaminatedmicroorganisms.

The expressed polypeptide and/or the polypeptide complex can be furtherpurified by a suitable method, such as without limitation, affinitychromatography, hydroxylapatite chromatography, size exclusionchromatography, gel electrophoresis, dialysis, ion exchangefractionation on an ion-exchange column, ethanol precipitation, reversephase HPLC, chromatography on silica, chromatography on heparinsepharose, chromatography on an anion or cation exchange resin (such asa polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation (see, for review, Bonner, P. L., Proteinpurification, published by Taylor & Francis, 2007; Janson, J. C., et al,Protein purification: principles, high resolution methods andapplications, published by Wiley-VCH, 1998).

In certain embodiments, the polypeptides and/or polypeptide dimercomplexes can be purified by affinity chromatography. In certainembodiments, protein A chromatography or protein A/G (fusion protein ofprotein A and protein G) chromatography can be useful for purificationof polypeptides and/or polypeptide complexes comprising a componentderived from antibody CH2 domain and/or CH3 domain (Lindmark et al., J.Immunol. Meth. 62:1-13 (1983)); Zettlit, K. A., Antibody Engineering,Part V, 531-535, 2010). In certain embodiments, protein G chromatographycan be useful for purification of polypeptides and/or polypeptidecomplexes comprising IgG γ3 heavy chain (Guss et al., EMBO J. 5:15671575 (1986)). In certain embodiments, protein L chromatography can beuseful for purification of polypeptides and/or polypeptide complexescomprising K light chain (Sudhir, P., Antigen engineering protocols,Chapter 26, published by Humana Press, 1995; Nilson, B. H. K. et al, J.Biol. Chem., 267, 2234-2239 (1992)). The matrix to which the affinityligand is attached is most often agarose, but other matrices areavailable. Mechanically stable matrices such as controlled pore glass orpoly(styrene-divinylbenzene) allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Compositions and Methods of Use

The present disclosure provides compositions comprising the MSFP asdescribed herein and administration of such composition in a variety oftherapeutic settings.

Administration of the MSFP described herein, in pure form or in anappropriate pharmaceutical composition, can be carried out via any ofthe accepted modes of administration of agents for serving similarutilities. The pharmaceutical compositions can be prepared by combiningan MSFP or an MSFP-containing composition with an appropriatephysiologically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. In addition, other pharmaceutically active ingredients(including other anti-cancer agents as described elsewhere herein)and/or suitable excipients such as salts, buffers and stabilizers may,but need not, be present within the composition. Administration may beachieved by a variety of different routes, including oral, parenteral,nasal, intravenous, intradermal, subcutaneous or topical. Preferredmodes of administration depend upon the nature of the condition to betreated or prevented. An amount that, following administration, reduces,inhibits, prevents or delays the progression and/or metastasis of acancer is considered effective.

In certain embodiments, the amount administered is sufficient to resultin tumor regression, as indicated by a statistically significantdecrease in the amount of viable tumor, for example, at least a 50%decrease in tumor mass, or by altered (e.g., decreased with statisticalsignificance) scan dimensions. In other embodiments, the amountadministered is sufficient to result in clinically relevant reduction indisease symptoms as would be known to the skilled clinician.

The precise dosage and duration of treatment is a function of thedisease being treated and may be determined empirically using knowntesting protocols or by testing the compositions in model systems knownin the art and extrapolating therefrom. Controlled clinical trials mayalso be performed. Dosages may also vary with the severity of thecondition to be alleviated. A pharmaceutical composition is generallyformulated and administered to exert a therapeutically useful effectwhile minimizing undesirable side effects. The composition may beadministered one time, or may be divided into a number of smaller dosesto be administered at intervals of time. For any particular subject,specific dosage regimens may be adjusted over time according to theindividual need.

The MSFP-containing compositions may be administered alone or incombination with other known cancer treatments, such as radiationtherapy, chemotherapy, transplantation, immunotherapy, hormone therapy,photodynamic therapy, etc. The compositions may also be administered incombination with antibiotics.

Typical routes of administering these and related pharmaceuticalcompositions thus include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Pharmaceutical compositions accordingto certain embodiments of the present invention are formulated so as toallow the active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a subject or patient may take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a herein described MSFP in aerosol form may hold aplurality of dosage units. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington: The Science and Practice of Pharmacy, 20thEdition (Philadelphia College of Pharmacy and Science, 2000). Thecomposition to be administered will, in any event, contain atherapeutically effective amount of an MSFP of the present disclosure,for treatment of a disease or condition of interest in accordance withteachings herein.

A pharmaceutical composition may be in the form of a solid or liquid. Inone embodiment, the carrier(s) are particulate, so that the compositionsare, for example, in tablet or powder form. The carrier(s) may beliquid, with the compositions being, for example, an oral oil,injectable liquid or an aerosol, which is useful in, for example,inhalatory administration. When intended for oral administration, thepharmaceutical composition is preferably in either solid or liquid form,where semi-solid, semi-liquid, suspension and gel forms are includedwithin the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like. Such a solid compositionwill typically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following may be present: binders such ascarboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gumtragacanth or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent. When thepharmaceutical composition is in the form of a capsule, for example, agelatin capsule, it may contain, in addition to materials of the abovetype, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions, whether they be solutions,suspensions or other like form, may include one or more of the followingadjuvants: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or diglycerides whichmay serve as the solvent or suspending medium, polyethylene glycols,glycerin, propylene glycol or other solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic. Physiological saline isa preferred adjuvant. An injectable pharmaceutical composition ispreferably sterile.

A liquid pharmaceutical composition intended for either parenteral ororal administration should contain an amount of an MSFP as hereindisclosed such that a suitable dosage will be obtained. Typically, thisamount is at least 0.01% of the MSFP in the composition. When intendedfor oral administration, this amount may be varied to be between 0.1 andabout 70% of the weight of the composition. Certain oral pharmaceuticalcompositions contain between about 4% and about 75% of the MSFP. Incertain embodiments, pharmaceutical compositions and preparationsaccording to the present invention are prepared so that a parenteraldosage unit contains between 0.01 to 10% by weight of the MSFP prior todilution.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. The pharmaceutical composition may beintended for rectal administration, in the form, for example, of asuppository, which will melt in the rectum and release the drug. Thecomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Such bases include, withoutlimitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition may include various materials, whichmodify the physical form of a solid or liquid dosage unit. For example,the composition may include materials that form a coating shell aroundthe active ingredients. The materials that form the coating shell aretypically inert, and may be selected from, for example, sugar, shellac,and other enteric coating agents. Alternatively, the active ingredientsmay be encased in a gelatin capsule. The pharmaceutical composition insolid or liquid form may include an agent that binds to the antibody ofthe invention and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include other monoclonalor polyclonal antibodies, one or more proteins or a liposome. Thepharmaceutical composition may consist essentially of dosage units thatcan be administered as an aerosol. The term aerosol is used to denote avariety of systems ranging from those of colloidal nature to systemsconsisting of pressurized packages. Delivery may be by a liquefied orcompressed gas or by a suitable pump system that dispenses the activeingredients. Aerosols may be delivered in single phase, bi-phasic, ortri-phasic systems in order to deliver the active ingredient(s).Delivery of the aerosol includes the necessary container, activators,valves, subcontainers, and the like, which together may form a kit. Oneof ordinary skill in the art, without undue experimentation maydetermine preferred aerosols.

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art. For example, a pharmaceuticalcomposition intended to be administered by injection can be prepared bycombining a composition that comprises an MSFP as described herein andoptionally, one or more of salts, buffers and/or stabilizers, withsterile, distilled water so as to form a solution. A surfactant may beadded to facilitate the formation of a homogeneous solution orsuspension. Surfactants are compounds that non-covalently interact withthe MSFP composition so as to facilitate dissolution or homogeneoussuspension of the MSFP in the aqueous delivery system.

The compositions may be administered in a therapeutically effectiveamount, which will vary depending upon a variety of factors includingthe activity of the specific compound (e.g., MSFP) employed; themetabolic stability and length of action of the compound; the age, bodyweight, general health, sex, and diet of the patient; the mode and timeof administration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy. Generally, a therapeutically effective daily dose is(for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (fora 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg(i.e., 3.5 g); more preferably a therapeutically effective dose is (fora 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg(i.e., 1.75 g).

Compositions comprising the MSFP of the present disclosure may also beadministered simultaneously with, prior to, or after administration ofone or more other therapeutic agents. Such combination therapy mayinclude administration of a single pharmaceutical dosage formulationwhich contains a compound of the invention and one or more additionalactive agents, as well as administration of compositions comprising MSFPof the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, an MSFP as describedherein and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Similarly, an MSFP as described herein and the otheractive agent can be administered to the patient together in a singleparenteral dosage composition such as in a saline solution or otherphysiologically acceptable solution, or each agent administered inseparate parenteral dosage formulations. Where separate dosageformulations are used, the compositions comprising MSFP and one or moreadditional active agents can be administered at essentially the sametime, i.e., concurrently, or at separately staggered times, i.e.,sequentially and in any order; combination therapy is understood toinclude all these regimens.

Thus, in certain embodiments, also contemplated is the administration ofMSFP compositions of this disclosure in combination with one or moreother therapeutic agents. Such therapeutic agents may be accepted in theart as a standard treatment for a particular disease state as describedherein, such as cancer, inflammatory disorders, allografttransplantation, type I diabetes, and multiple sclerosis. Exemplarytherapeutic agents contemplated include cytokines, growth factors,steroids, NSAIDs, DMARDs, anti-inflammatories, chemotherapeutics,radiotherapeutics, or other active and ancillary agents.

In certain embodiments, the MSFP disclosed herein may be administered inconjunction with any number of chemotherapeutic agents. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide-K(PSK™); razoxane; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2, 2′,2″-trichlorotriethylamine; urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids,e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid derivatives such asTARGRETIN™ (bexarotene), PARETIN™ (alitretinoin); ONTAK™ (denileukindiftitox); esperamicins; capecitabine; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. Also included in thisdefinition are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (FARESTON®); and anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

A variety of other therapeutic agents may be used in conjunction withthe MSFP described herein. In one embodiment, the MSFP is administeredwith an anti-inflammatory agent. Anti-inflammatory agents or drugsinclude, but are not limited to, steroids and glucocorticoids (includingbetamethasone, budesonide, dexamethasone, hydrocortisone acetate,hydrocortisone, hydrocortisone, methylprednisolone, prednisolone,prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs(NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate,sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide andmycophenolate.

The compositions comprising herein described MSFP may be administered toan individual afflicted with a disease as described herein, including,but not limited to cancer and autoimmune and inflammatory diseases. Forin vivo use for the treatment of human disease, the MSFP describedherein are generally incorporated into a pharmaceutical compositionprior to administration. A pharmaceutical composition comprises one ormore of the MSFP described herein in combination with a physiologicallyacceptable carrier or excipient as described elsewhere herein. Toprepare a pharmaceutical composition, an effective amount of one or moreof the MSFPs is mixed with any pharmaceutical carrier(s) or excipientknown to those skilled in the art to be suitable for the particular modeof administration. A pharmaceutical carrier may be liquid, semi-liquidor solid. Solutions or suspensions used for parenteral, intradermal,subcutaneous or topical application may include, for example, a sterilediluent (such as water), saline solution, fixed oil, polyethyleneglycol, glycerin, propylene glycol or other synthetic solvent;antimicrobial agents (such as benzyl alcohol and methyl parabens,phenols or cresols, mercurials, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride); antioxidants (such as ascorbic acid and sodiumbisulfite; methionine, sodium thiosulfate, platinum, catalase, citricacid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylatedhydroxanisol, butylated hydroxytoluene, and/or propyl gallate) andchelating agents (such as ethylenediaminetetraacetic acid (EDTA));buffers (such as acetates, citrates and phosphates). If administeredintravenously, suitable carriers include physiological saline orphosphate buffered saline (PBS), and solutions containing thickening andsolubilizing agents, such as glucose, polyethylene glycol, polypropyleneglycol and mixtures thereof.

The compositions comprising MSFP as described herein may be preparedwith carriers that protect the MSFP against rapid elimination from thebody, such as time release formulations or coatings. Such carriersinclude controlled release formulations, such as, but not limited to,implants and microencapsulated delivery systems, and biodegradable,biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, polyorthoesters, polylactic acid and others known tothose of ordinary skill in the art.

The present MSFP are useful for the treatment of a variety of cancers.For example, one embodiment of the invention provides a method for thetreatment of a cancer including, but not limited to, melanoma,non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma,sarcoma, glioma, thymoma, breast cancer, prostate cancer, colo-rectalcancer, kidney cancer, renal cell carcinoma, uterine cancer, pancreaticcancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer,cervical cancer, testicular cancer, gastric cancer, esophageal cancer,multiple myeloma, hepatoma, acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), andchronic lymphocytic leukemia (CLL), or other cancers, by administeringto a cancer patient a therapeutically effective amount of a hereindisclosed MSFP. An amount that, following administration, inhibits,prevents or delays the progression and/or metastasis of a cancer in astatistically significant manner (i.e., relative to an appropriatecontrol as will be known to those skilled in the art) is consideredeffective.

Another embodiment provides a method for preventing metastasis of acancer including, but not limited to, melanoma, non-Hodgkin's lymphoma,Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma,breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renalcell carcinoma, uterine cancer, pancreatic cancer, esophageal cancer,brain cancer, lung cancer, ovarian cancer, cervical cancer, testicularcancer, gastric cancer, esophageal cancer, multiple myeloma, hepatoma,acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia(CLL), or other cancers, by administering to a cancer patient atherapeutically effective amount of a herein disclosed MSFP (e.g., anamount that, following administration, inhibits, prevents or delaysmetastasis of a cancer in a statistically significant manner, i.e.,relative to an appropriate control as will be known to those skilled inthe art).

Another embodiment provides a method for preventing a cancer including,but not limited to, melanoma, non-Hodgkin's lymphoma, Hodgkin's disease,leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer,prostate cancer, colo-rectal cancer, kidney cancer, renal cellcarcinoma, uterine cancer, pancreatic cancer, esophageal cancer, braincancer, lung cancer, ovarian cancer, cervical cancer, testicular cancer,gastric cancer, esophageal cancer, multiple myeloma, hepatoma, acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), orother cancers, by administering to a cancer patient a therapeuticallyeffective amount of a herein disclosed MSFP.

Another embodiment provides a method for treating, inhibiting theprogression of or prevention of melanoma, non-Hodgkin's lymphoma,Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma,breast cancer, prostate cancer, colo-rectal cancer, kidney cancer, renalcell carcinoma, uterine cancer, pancreatic cancer, esophageal cancer,brain cancer, lung cancer, ovarian cancer, cervical cancer, testicularcancer, gastric cancer, esophageal cancer, multiple myeloma, hepatoma,acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia(CLL), or other cancers, by administering to a patient afflicted by oneor more of these diseases a therapeutically effective amount of a hereindisclosed MSFP.

In one aspect, the present disclosure provides a method for directing Tcell activation, comprising administering to a patient in need thereofan effective amount of an MSFP that comprises a Fab that specificallybinds TCRα, TCRβ, CD3γ, CD3δ, CD3ε or a combination thereof, and afusion moiety the comprises a binding domain that specifically binds adifferent target, for instance, a tumor-specific antigen or otherantigen of choice at a site or cell where T cell activation is desired.

EXAMPLES Example 1: Generation of Humanized Anti-CD3 Antibodies

The heavy and light chain antibody genes were cloned from an establishedanti-CD3 hybridoma by steps 1) isolation of total RNA from hybridomacells, 2) reverse transcription (RD of the hybridoma total RNA and 3)PCR amplification of HC and LC using specific antibody gene primers. RTPCR was performed using M-muLV reverse transcriptase (New EnglandBiolabs) with oligo-dT₁₆ primer for 2 hrs at 37° C. The antibody heavychain and light chain genes were PCR amplified from the oligo-dT₁₆primed first strand cDNA using primer pairs of HC5′: GAG ACA GAATTCGCCACC ATG GTG TTG GGG CTG AAG TG (SEQ ID NO: 103), HC3′: GAG ACAGCGGCCGC CTA TTT ACC AGG GGA GCG AGA C (SEQ ID NO: 104) and LC5′: GAGACA GAATTC GCCACC ATG GCC TGG ATT TCA CTT ATA C (SEQ ID NO: 105), LC3′:GAG ACA GCGGCCGC TCA GGA ACA GTC AGC ACG GGA C (SEQ ID NO: 106). The 5′primers were designed to anneal to the antibody secretion signal and the3′ primers were designed to anneal to the 3′ end of the mouse antibodyconstant region. PCR products were cleaned and restriction digested withEcoRI and NotI and subsequently cloned into pcDNA3.3 vector for DNAsequence determination and recombinant expression of IgG in a 293Ftransient mammalian system. The cloned antiCD3 antibody is referred toas 1F3. The amino acid sequences for the 1F3 VHCDR1, CDR2 and CDR3 areprovided in SEQ ID NOs: 23-25. The amino acid sequences for the 1 F3VLCDR1, CDR2 and CDR3 are provided in SEQ ID NOs: 26-28.

Conventional CDR grafting technique was used for humanization of the 1F3mouse antibody. In particular, the amino acid sequences of the mouse 1F3were aligned with human germline sequences (VBASE2, at the world-wideweb address vbase2.org). Mouse 1F3VH matched best to the human germlinesegments subclass 3 (VH3). Two representative germline segments, namelyVH3-23 and VH3-73 were chosen as the acceptor on to which the mouse 1F3CDRs were grafted. VH3-23 is known to be the most frequent V domainfound in human antibodies and it has relatively high stability. VH3-73has the most similar HCDR2 sequence suggesting similar HCDR2 structureand conformation. For VL, the best matched human germline, VLλ7a waschosen as the acceptor for mouse 1F3 light chain CDRs. The genes for theinitial humanized constructs were synthesized by GBLOCK® technology(Integrated DNA Technologies). Further variation of amino acids in theinitial humanization constructs were generated using oligonucleotidebased site-directed mutagenesis (Strategene).

Table 1 below summarizes the nucleotide and amino acid sequences for thevarious heavy and light chain variable regions generated.

TABLE 1 1F3 and Variant Sequences Nucleotide Amino acid Construct NameSEQ ID NO SEQ ID NO mu-1F3 VHCDR1 23 mu-1F3 VHCDR2 24 mu-1F3 VHCDR3 25mu-1F3 VLCDR1 26 mu-1F3 VLCDR2 27 mu-1F3 VLCDR3 28 hu-1F3-IgGHC 29 30hu-1F3LC 31 32 hu-1F3-1VH 33 34 hu-1F3-1Fd 35 36 hu-1F3-2VH 37 38hu-1F3-2Fd 39 40 hu-1F3-3VH 41 42 hu-1F3-3Fd 43 44 hu-1F3-4VH 45 46hu-1F3-4Fd 47 48 hu-1F3-5VH 49 50 hu-1F3-5Fd 51 52 hu-1F3-11VH 53 54hu-1F3-1VL 55 56 hu-1F3-2VL 57 58 hu-1F3-2LC 59 60 hu-1F3-3VL 61 62hu-1F3-3LC 63 64 hu-1F3-4VL 65 66 hu-1F3-4LC 67 68 hu-1F3-5VL 69 70hu-1F3-5LC 71 72 OKT3Fd 73 74 OKT3LC 75 76

A variety of humanized antibodies/Fabs were generated using differenthumanized 1F3 VL and VH pairs as summarized in Table 2 below.

TABLE 2 hu-1F3 IgG and Fab variants Humanized Humanized VL VH hu-1F3-1VLhu-1F3-2VL hu-1F3-1VH hu-1F3.1, hu-1F3.6 hu-1F3 IgG hu-1F3-2VH hu-1F3.2hu-1F3.7 hu-1F3-3VH hu-1F3.3 hu-1F3.8 hu-1F3-4VH hu-1F3.4 hu-1F3.9hu-1F3-5VH hu-1F3.5 hu-1F3.10 hu-1F3-11VH

Example 2: Tumor Antigen EpCAM Protein Generation

The genes corresponding to the extracellular domain of human andcynomolgus EpCAM (epithelial cell adhesion molecules) was PCR amplifiedfrom human and monkey cDNA libraries using primers with restrictionsites (SEQ ID NO: 107: N-term primer: GCGTAT CCATGG ATG GCG CCC CCG CAGGTC, SEQ ID NO: 108: C-term primer:

GCGTAT GCGGCCGC TTT TAG ACC CTG CAT TGA G)with PHUSION® polymerase (New England Biolabs) (98° C., 1 min; 30 cyclesof 98° C. for 15 s, 50° C. for 20 s and 72° C. for 20 s; 72° C., 5 min).Cleaned PCR products (Fermentas life sciences) were restriction digested(NcoI and NotI) and cloned into the N-terminus of a human Fc gene in apcDNA3.3 vector. Human EpCAM ECD and full-length polynucleotidesequences are provided in SEQ ID NOs: 1 and 5; encoding the amino acidsequences provided in SEQ ID NOs: 2 and 6. Cynomolgus EpCAM ECD andfull-length polynucleotide sequences are provided in SEQ ID NOs: 3 and7; encoding the amino acid sequences provided in SEQ ID NOs: 4 and 8.Human and cynomolgus CD3 epsilon ECD.Fc knob mutant sequences were alsogenerated and are provided in SEQ ID NOs: 9 and 11 respectively(polynucleotide) and 10 and 12, respectively (amino acid). Human andcynomolgus CD3 epsilon ECD.Fc hole mutant sequences were generated andare provided in SEQ ID NOs: 13 and 15, respectively (polynucleotide) andSEQ ID NOs: 14 and 16, respectively (amino acid). As is known in theart, the knob and whole mutants can be used for formingheterodimerization (see e.g., Ridgeway et al. (1996), proteinEngineering 9:617-621).

Example 3: Generation of Anti-EPCAM Antibodies Using Human ScFv PhageDisplay

General protocols are referenced in Phage display technology—alaboratory manual (Eds Carlos F. Barbas III; Dennis R. Burton; Jamie K.Scott; Gregg J. Silverman, 2001 Cold Spring Harbor Laboratory Press). Anaïve human scFv phage display library was used for isolation of EpCAMspecific antibodies (Viva Biotech, Shanghai, China). The following stepswere employed for round 1 phage selection: 1) 50 ul of human naïve ScFvlibrary was blocked with 500 ul 3% non-fat dry milk/PBS (MPBS); 2)Incubate milk blocked phage with immunotube pre coated with 5 μg/ml Fcfor 30 minutes at room temperature, repeated 2 times; 3) add Fc proteinto the above depleted phage at the final concentration of 500 μg/ml andincubated at room temperature to further capture phage displayinganti-Fc ScFv; 4) incubate the above phage solution with huEpCAM.Fccoated immuno-tube(coated with a 5 μg/ml concentration and blocked withMPBS) at room temperature 1 hr; 5) the tube was washed 3 times with PBST(PBS containing 0.1% TWEEN®-20 (i.e., polysorbate 20)) followed by 3washes with PBS; 6) The bound phage was eluted with 500 μl of freshlyprepared solution of 100 mM triethylamine for 10 minutes at roomtemperature; 7) The eluted phage was used to infect 5 ml of mid logphase TG1 cells at 37° C. for 30 min stationary and 30 min with shakingat 200 rpm; 8) TG1 cells reinfected with eluted phage were grown at 30°C. overnight on large square NUNC® plates of 2×YT agar supplemented with4% glucose and 100 μg/ml ampicillin; 9) overnight grown TG1 cells werescraped off the plate and inoculated to 25 ml of 2×YT media (plus 100ug/ml ampicillin and 2% glucose and grown at 37° C. till OD600; 10)helper phage KO7 was added to the TG1 culture to rescue the phage (30min stationary, 30 minutes shaking at 37° C.); 11) Superinfected TG1cells were gently pelleted and resuspended into 2×YT media supplementedwith 100 μg/ml ampicillin and 100 μg/ml kanamycin to shake at 37° C.overnight; 12) Overnight culture was centrifuged and ⅕ volume of 20%PEG8000 was added to the cleared supernatant containing phage andincubated at room temperature for 30 min; 13) the precipitated phage waspelleted by centrifugation in 50 ml conical tubes at 3000 rpm for 20minutes; 14) pelleted phage was resuspended into PBS buffer and is readyfor use in next round of selection.

Round 2 selection was done on cynoEpCAM.Fc using the same stepsdescribed above. The eluted phage from round 2 selection wasreintroduced into TG1 cells and plated for single colonies on 2×YT (100ug/ml ampicillin and 2% glucose) plates. Single colonies were pickedinto 96-well plates and duplicate plates were made as master plates forsamples. Individual clonal phage in 96 well plates was grown usingsimilar step described above adjusted for smaller volume. Cleared phagesupernatant (cleared by centrifugation of 96 well plates at 3000 rpm for5 minutes) was used for ELISA binding assay on human and cyno EpCAM.Fcantigens using the following steps: 1) phage solution was diluted at 1:5in 5% MPBS incubated at room temperature for 1 hr; 2) phage MPBSsolution was transferred to 96 well MaxiSorp® plates pre-coated withhuEpCAM.Fc or cyno EpCAM.Fc with blocking using MPBS; 3) plates wereincubated at room temperature for 1 hr and followed by 3 washes usingPBST; 4) 100 μl of 1/1000 diluted anti M13/HRP conjugate was added tothe plates and incubated at room temperature for 1 hr; 5) after 3 washeswith PBST, 100 μl of TMB peroxidase substrate solution was added toplates and incubated in dark for 20 minutes; 6) 50 μl of 0.25 Msulphuric acid was added to each well on the plate to stop substratedevelopment; 7) absorbance were read at 450 nm on a microplate reader.The EpCAM positive binders were DNA sequenced using E coli stock fromthe master plates.

Example 4: Optimization of Fully Human Anti-EpCAM Antibodies

The VH and VL genes of anti EpCAM antibodies were synthesized usingGBLOCK® technology (Integrated DNA Technologies). The V gene GBLOCKS®were either identical to those isolated form phage display technology orcontained mutations designed to improve the expression and/or stabilityof the V gene products. The design of mutations is primarily based onthe sequence comparison with the best matched germline sequence orconsensus of the germlines (VBASE2 at the world-wide web addressvbase2.org) or by examining the homologous V domain structures. The VHand VL GBLOCKS® were assembled into the scFv format using PCRmethodology. For example, EpCAM1.1 contains amino acid changes of VLresidue 1-3 (Kabat numbering, Kabat et al. (1991), sequences of proteinsof immunological interest, 5th edition) from “HVI” in EpCAM1.0 to “QSV”.The latter is common according to human germline sequences (VBASE2, atthe world-wide web address vbase2.org). EpCAM1.2 contains mutations atposition 1, 5, 6 in VH and position 39 in VL (Kabat numbering) incomparison to EpCAM1.1. EpCAM2.2 contains multiple mutations compared tothe parental clone at position 5, 6, 13, 40, 76, 77, 81, 82 in VH andpositions 2, 8, 39, 58 in VL (Kabat numbering). The anti EpCAM antibodynucleotide and amino acid sequences are summarized in Table 3 below.

TABLE 3 Anti-EpCAM antibody constructs Nucleotide Amino acid ConstructName SEQ ID NO SEQ ID NO EpCAM1.1scFv 77 78 EpCAM1.1-OKT3Fd 79 80EpCAM1.1-OKT3LC 81 82 EpCAM1.1-hu-1F3.1Fd 83 84 EpCAM1.1-hu-1F3.1LC 8586 EpCAM1.2scFv 87 88 EpCAM1.2-hu-1F3Fd 89 90 EPCAM1.2-hu-1F3.1LC 91 92EpCAM2.2scFv 93 94 EpCAM2.2-hu-1F3Fd 95 96 EpCAM2.2-hu-1F3.1LC 97 98EpCAM2.2-Δ-hu-1F3.1Fd* 99 100 EpCAM2.2-Δ-hu-1F3.1LC* 101 102 EpCAM1.1VHCDR1 139 EpCAM1.1 VHCDR2 140 EpCAM1.1 VHCDR3 141 EpCAM1.1 VLCDR1 142EpCAM1.1 VLCDR2 143 EpCAM1.1 VLCDR3 144 EpCAM1.2 VHCDR1 139 EpCAM1.2VHCDR2 140 EpCAM1.2 VHCDR3 141 EpCAM1.2 VLCDR1 142 EpCAM1.2 VLCDR2 143EpCAM1.2 VLCDR3 144 EpCAM2.2 VHCDR1 145 EpCAM2.2 VHCDR2 146 EpCAM2.2VHCDR3 147 EpCAM2.2 VLCDR1 148 EpCAM2.2 VLCDR2 149 EpCAM2.2 VLCDR3 150*Δ indicates no linker

A variety of MSFP proteins were generated using the anti EpCAM and hu-1F3.1 constructs, as summarized below. scFv in the summary below refersto an anti-EpCAM scFv. His-tag is attached to the C-terminus of the Fdchain in each Fab and is not shown in the illustration.

scFv-D(H)-hu-1F3.1 Fab scFv-hu-1F3.1VH-CH1 hu-1F3.1-LC scFv-(H)-hu-1F3.1Fab scFv-GlyArgAla-hu-1F3.1VH-CH1 hu-1F3.1-LC scFv-D(L)-hu-1F3.1 Fabhu-1F3.1VH-CH1 scFv-hu-1F3.1-LC scFv-(L)-hu-1F3.1 Fab hu-1F3.1VH-CH1scFv-GlyArgAla-hu-1F3.1-LC scFv-D(H + L)-hu-1F3.1 FabscFv-hu-1F3.1VH-CH1 scFvhu-1F3.1-LC scFv-(H + L)-hu-1F3.1 FabscFv-GlyArgAla-hu-1F3.1VH-CH1 scFv-GlyArgAla-hu-1F3.1-LC

Example 5: Recombinant IgG, Fc Fusion Protein, Fab, and Fab FusionProtein Expression and Purification

Fab fusion proteins were expressed in a transient mammalian expressionsystem employing pcDNA and HEK293F suspension cell (Invitrogen). Theexpression constructs were transfected into HEK293F cells (Invitrogen)adding preformed DNA and 25 kD polyethylenimine (PEI) complex (DNA tolinear 25 kD PEI at 1:3 ratio by weight) in 1/10 of cell culture volumeof F-17 synthetic medium (Invitrogen). Transfected cells grown in 5%moisturized CO2 incubator with shaking were fed with 25 ml of 20% TN1(Organotechnie SA, France) 24 hr post-transfection. Culture supernatantswere usually harvested 5 days post-transfection and proteins werepurified using affinity chromatography by either protein A (for humanIgG and Fc proteins), protein G (for mouse IgG), or HISTRAP® columns(for his-tagged Fab and his-tagged Fab fusions) (GE Healthcare). Afterbuffer exchange using 10 kD MW cut off spin tubes, proteins were storedin PBS buffer at 4° C. Proteins were usually analyzed by 4-20% SDS-PAGE(polyacrylamide gel electrophoresis, NOVEX® mini gel) under non-reducingand/or reducing condition (5% mercaptoethanol).

Example 6: Mouse 1 F3 and Humanized 1F3 IgG and Fab Binding to CD3Epsilon Related Proteins

Western blot, ELISA and flow cytometric analysis were used to studybinding of mouse and human IF3 antibodies and Fabs. In particular, thefollowing four protein samples were prepared in an SDS-PAGE samplebuffer with 5% mercaptoethanol (samples 1. humanCD3epsilon/delta.Fc(K-H); 2. cyno CD3epsilon/delta.Fc(K-H); 3). humanCD3epsilonAA1-27.Fc; and 4) control peptide.Fc fusion (reverse aminoacid sequence of the first 16 residues in CD3epsilonAA1-27.Fc. “Fc(K-H)”denotes “knob and hole” Fc mutants (Ridgeway et al. (1996), proteinEngineering 9:617-621). 100 ng of protein for each sample was loaded toeach lane on a 4-20% tris-glycine gel (Novex) and samples were run in1×SDS running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH8.3) at125V for 1 hr. Protein bands were transferred to a nitrocellulose (NC)membrane in 1×SDS running buffer with 20% methanol using a NOVEX®transfer module. NC Membranes were blocked with 5% dry milk TBS-Tfollowed by incubation with 1 μg/ml of biotinylated mouse 1F3 IgG orbiotinylated hu-1 F3 IgG for 1 hr at rt. After 3×5 min washes, themembranes were incubated with 1:3000 diluted streptavidin-HRP conjugatefor 1 hr. After 3×10 minute washes, the membrane was developed ECLreagent and signals exposed to Kodak films.

As shown in FIG. 9, the mouse 1F3 IgG and the humanized version of 1F3recognized this panel of antigen in a very similar manner.Specifically, 1) both mouse 1F3 and the hu-1F3 IgG are able to binddenatured human CD3epsilon/delta and the cynomolgus CD3epsilon/delta; 2)both antibodies are able to bind denatured Fc fusion protein containingthe peptide sequence corresponding to the N-terminal amino acid 1 to 27of CD3epsilon (SEQ ID NO: 18); and 3) neither mouse 1F3 nor hu-1F3 IgGbinds to a Fc fusion protein with a control peptide (SEQ ID NO: 20).Results from this experiment strongly suggest that 1F3 is specific toCD3epsilon and the epitope is within the N-terminal part of the epsilonsubunit. Flow cytometric analysis also showed a similar binding patternon human PBMCs for the human and mouse 1F3 antibodies (see FIG. 10). Ofnote, a small shift in the MFI of the low fluorescent cell populationswas observed. It is well known that mouse Fc has much reduced binding tohuman Fc gamma receptors compared to the human Fc. Thus, the resultsshown in FIG. 10 suggest that human Fc in the hu-1F3 IgG binds to theFcgamma receptors on this population of immune cells (non T cells) inthe PBMC prep and caused the small MFI shift.

MAXISORP® plates were used for ELISA binding assays. 50 μl of 1 μg/ml ofantigens in 50 mM NaHCO₃were used to coat the plates at 4° C. overnightfollowed by incubation with 200 μl of 5% dry milk/TBS-0.05% TWEEN®-20(i.e., polysorbate 20) (MTBS-T)/well. Antibodies were usually diluted in5% MTBS-T and transferred to antigen coated plates and incubated for 1hr with gentle shaking at room temperature. Bound antibodies weredetected either by streptavidin-HRP (for detecting biotinylatedantibodies) or anti-his tag-HRP conjugate (for his tagged antibodies).ABTS™ (i.e., 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)solution in 50 mM citrate buffer was used for detection with OD405measurement on using a microplate reader (Biotek).

FIG. 11 shows the binding activities of biotinylated mouse antiCD3antibody 1F3 and biotinylated hu-1F3 IgG. The two antibodiesdemonstrated very similar pattern of positive binding to humanCD3epsilon/delta “knob and hole” Fc heterodimer (Ridgeway et al. (1996),protein Engineering 9:617-621), to cynomolgus CD3epsilon/delta “knob andhole” Fc heterodimer and to the human CD3epsilon N-terminal peptide(AA1-27) Fc fusion protein.

FIG. 13 shows binding to the same panel of antigens described in FIG. 11by ten representative humanized Fab proteins detected with anti histag/HRP conjugate and ABTS substrate. Dose dependent binding wasobserved for hu-1F3.1, hu-1F3.2, hu-1F3.3 and hu-1F3.5 through hu-1F3.10Fabs. Hu-1F3.4 Fab binding is very low even in the concentration rangetested (5-8,000 pM). Slightly lower affinities for the Fab binding toall three antigens were noticed as compared to the IgG (FIG. 11).Monovalent binding of Fab may explain the lower binding affinitycompared to that of the hu-1F3 full IgG (in FIG. 11).

Five of the ten representative humanized Fab proteins (hu-IF3.1-huIF3.5)were tested for binding to the Jurkat human T-cell line expressing CD3.As shown in FIG. 12, the humanized 1F3 Fabs bind to Jurkat cells.

Example 7: Characterization of the EpCAM×Cd3 Bispecific Fab FusionProteins

Flow cytometric analysis and ELISA were used to characterize theEpCAM×CD3 Fab fusion proteins. Detailed materials and methods aredescribed at the end of this example.

As shown in FIG. 14, EpCAM×CD3 Fab fusion proteins (MSFP) bind to Jurkatcells expressing human CD3 using FACS. In this experiment, biotinylatedFab and Fab fusions bound to Jurkat cells were detected bystreptavidin-PE conjugate. OKT3 Fab showed binding to CD3 on Jurkatcells but the anti EpCAM scFv fusion to either the LC alone or to bothHC and LC completely abolished the CD3 binding ability of OKT3 Fabmoiety. Humanized antiCD3 antibody Fab, hu-1 F3 was able to bind CD3 onJurkat cells. Contrary to OKT3 Fab fusion proteins, anti EpCAM scFvfused to the N-terminal of hu-1 F3 LC retained the binding activity atsimilar level as the hu-1F3 Fab; simultaneous fusion of anti EpCAM scFvto the LC and HC of hu-1F3 Fab also showed positive binding to CD3 onJurkat cells, though at a reduced level.

FIG. 15 shows EpCAM×CD3 MSFP binding to human PBMCs as shown by FACS.Biotinylated Fab and Fab fusions bound to Jurkat cells were detected bystreptavidin-PE conjugate. Panel A shows OKT3 Fab bound to PBMCsexpressing CD3 (T cells); the anti EpCAM scFv to both HC and LCcompletely abolished the CD3 binding ability of OKT3 Fab moiety. Panel Bshows humanized antiCD3 antibody Fab, hu-1F3.1 is able to bind CD3 on Tcells in PBMC preparation; unlike the OKT3 Fab fusion protein, antiEpCAM×hu-1F3.1 Fab with simultaneous fusions to the LC and HC of Fabshowed positive binding to CD3 on Jurkat cells and the level of bindingis at a reduced level.

As shown in FIG. 16, ELISA binding assays showed that: OKT3 Fab andEpCAM1.1×OKT3 bispecific Fab fusion proteins have no binding activitytowards the recombinant cyno CD3epsilon/delta heterodimeric Fc protein;and OKT3 Fab and bispecific fusions have no binding activity towards therecombinant human CD3epsilon N-terminal peptide (aa1-27.Fc fusion). Itis expected that OKT3 does not bind to monkey CD3 or the N-terminus ofhuman CD3 epsilon. Fab and Fab fusion proteins were biotinylated and thebiotin-antibodies bound to 96-well plate immobilized antigens weredetected using streptavidin-HRP conjugate followed by ABTS substrate fordetection.

FIG. 17 shows ELISA binding results demonstrating that hu-1F3 Fab andEpCAM1.1×hu-1F3.1 bispecific Fab fusion proteins (MSFP; Fabe) bind therecombinant cyno CD3epsilon/delta heterodimeric Fc protein (panel A);and the recombinant human CD3epsilon N-terminal peptide (aa1-27.Fcfusion) (panel B). Fab and Fab fusion proteins were biotinylated and thebiotin-antibodies bound to 96-well plate immobilized antigens weredetected using streptavidin-HRP conjugate followed by ABTS substrate fordetection.

As shown in FIG. 18, ELISA binding assays showed that hu-1F3 Fab andEpCAM1.2×hu-1F3.1 MSFP bind the recombinant cyno CD3epsilon/deltaheterodimeric Fc protein (panel A); and towards the recombinant humanCD3epsilon N-terminal peptide (aa1-27.Fc fusion) (panel B). Fab and Fabfusion proteins were biotinylated and the labeled fusion proteins boundto 96-well plate immobilized antigens were detected usingstreptavidin-HRP conjugate followed by ABTS substrate for detection.

As shown in FIG. 19, ELISA binding assays showed that hu-1F3.1 Fab andEpCAM2.2×hu-1F3 MSFP bind the recombinant cyno CD3epsilon/deltaheterodimeric Fc protein (panel A); and towards the recombinant humanCD3epsilon N-terminal peptide (aa1-27.Fc fusion) (panel B). Fab and Fabfusion proteins were biotinylated and the biotin-labeled fusion proteinsbound to 96-well plate immobilized antigens were detected usingstreptavidin-HRP conjugate followed by ABTS substrate for detection.

As shown in FIG. 20, flow cytometric analysis showed EpCAM×CD3 MSFPbinding to CHO cells stably expressing full length human EpCAM target onthe cell surface. Anti CD3 Fab showed no binding to the same CHO cells.

Materials and Methods: Flow cytometric analysis (Fluorescence activatedcell sorting; FACS) was used to characterize the cell binding ofIF3-based Fab fusion proteins (also referred to herein as a Fabe; Fabfusion proteins are also generically referred to as MSFP). In general,cells were blocked in 1% BSA/PBS for 1 hr at 4 C and antibodies ofinterest were diluted into 1% BSA/PBS were added to blocked cells forincubation at 4° C. for 1 hr. after one wash with 1% BSA/PBS, stainingprocedures vary depending on the availability of antibodies orfluorescent antibody conjugate. In some case, direct fluorescenceconjugates were used. In other cases, anti affinity tag antibodies(secondary antibody) were used to incubate with the cells followed byanti-species antibody conjugated with a particular fluorophore. In someother experiments, antibody (or other affinity reagent such asstreptavidin) with fluorescence conjugate was used directly as secondaryantibody (secondary reagent). In FIGS. 10, 14, 16, 17, and 20 (panels Aand B) biotinylated antibodies were used to incubate with cells and inFIGS. 12, 15, 18, 19, and 20 (panels C and D), his6 tagged antibodieswere used for incubation with cells followed by anti his tag antibodyand final detection is by anti-mouse PE-conjugate.

Example 8: EpCAM×CD3 Bispecific Fab Fusion Proteins Redirect T-CellMediated Cell Killing Activity

A cell killing assay was used to test the ability of the EpCAM×CD3bispecific Fab fusion proteins to redirect T-cell mediated killing oftarget cells.

As shown in FIG. 21, a FACS based assay (described in detail at the endof the Example) demonstrated that the EpCAM×CD3 bispecific Fab fusionproteins redirect T cell-mediated target cell killing activity in atumor target dependent manner. In this assay, target cells (CHO cellsstably expressing human EpCAM-FL on cell surface (FIG. 21, panels A, B,and D) and control cells (CHO only, FIG. 21 panel C) were labeled withPKH-26 fluorescence dye prior to the assay. TP3 (Invitrogen) was used toidentify dead cells upon completion of the assay. In this assay, deadtarget cells were identified from the counts in the upper-right quadrantand the live target cells were identified from the upper-left quadrant.EpCAM expressing CHO cells incubated (for 20 hrs) without PBMC andbispecific antibody showed ˜1.4% dead cells among the PKH-26 labelledpopulations (TP3 positive) (FIG. 21, panel A); EpCAM-expressing CHO cellincubated (for 20 hrs) with PBMCs but without bispecific antibody had˜14% dead cells among the PKH-26 labelled populations (FIG. 21 Panel B);non EpCAM-expressing CHO cells incubated (for 20 hrs) with PBMCs andbispecific antibody had ˜14% dead cells (FIG. 21 Panel C); andEpCAM-expressing CHO cells incubated (for 20 hrs) with PBMCs andexemplary bispecific Fab fusions had ˜64% dead cell counts (FIG. 21,panel D).

FIG. 22 plots the killing data as described above in FIG. 21, Panel D,for various concentrations of EpCAM×CD3 bispecific Fab fusions. OKT3 Faband fusion proteins lack significant redirected cytolytic activity (FIG.22, panel A); EpCAM1.1×hu-1F3.1 MSFP had high level activity and theactivity level remained high even at 60 pM fusion protein concentration,while hu-1F3.1 Fab had no activity towards target cells (FIG. 22, panelB); dose dependent activities were detected by EpCAM1.2×hu-1F3.1 MSFP(FIG. 22, Panel C); and dose dependent cytolytic activities weredetected for EpCAM2.2 scFv single fusions to hu-1F3.1 Fab (FIG. 22,Panel D); for double fusion of EpCAM2.2 scFv to both the HC and LC ofhu-1F3.1 Fab, a maximum killing of 50% cell population were observed andthe activity level remained high (˜40%) for concentrations as low as 60pM. Percentage killing activities were calculated by subtractingpercentage of dead cells in the control assay (EpCAM-CHO+PBMC) from thepercentage of dead cells in the sample assay.

FIG. 23 shows that T cell activation by EpCAM2.2-(H+L)-hu-1F3.1 MSFP istumor target dependent. Panel A) PBMCs incubated withEpCAM2.2-(H+L)-hu-1F3.1(30 nM) in the presence of non EpCAM-expressingCHO resulted in basal level T cell activation measured by CD69expression detected by FACS assay; Panel B) PBMCs incubated withEpCAM2.2-(H+L)-hu-1F3.1(30 nM, 16 hrs) in the presence of nonEpCAM-expressing CHO resulted in significant increase of T cellactivation measured by CD69 expression detected by FACS assay.

Materials and Methods: Target cells were labeled with PKH-26 dye (Sigma)(signal detection in FL2) prior to the assay and killed target cells inthe assay were identified by addition of TO-PRO®-3 (TP3, i.e. acarbocyanine monomer nucleic acid stain) dye (Invitrogen) (signaldetection in FL4). PKH-26 labelling of target cells was performedaccording to manufacturers instructions. In particular, 2 million targetcells washed with serum free complete medium were resuspended in 0.5 mlof Diluent C (provided in the product kit). 2 μl of 1 mM PKH-26 dye wasalso diluted in 0.5 ml of Diluent C. The diluted dye was then added tothe target cell preparation and incubated at room temperature for 5 min.2 ml of fetal bovine serum was added to the mixture and incubated for 2minutes to stop the labeling reaction. Labeled cells were washed withcomplete media for three times and resuspended in complete media foruse. Labeled cells were counted and checked for viability. For cellkilling assay, 100 μl mixture of target cells and PBMCs in completemedia were added to FACS tubes followed by addition of 100 μl ofantibody solution in complete cell culture media and incubated for 20hrs in a CO2 incubator. At the end of the assay, 5 μl of 10 uM TO-PRO®3(TP3, i.e. a carbocyanine monomer nucleic acid stain) was added cellsimmediately before the FACS assay on a FACSCALIBUR™ flow cytometer.PKH-26 labelled cells were put in 2-3^(rd) log region (FL2). Cells withFL2 signal greater than log 20 were considered as target cells. Cellswith FL4 signal (TP3) greater than log 30 were considered as dead cells.Killed target cells were counted from the upper right quadrant and livetarget cells were counted from the upper left quadrant (see FIG. 21). Atotal of 5,000 target cell events were collected for each sample.Activity of cell lysis was calculated by subtracting the dead cellcounts of the control assay (EpCAM-expressing target cells plus PBMCs)from the dead cell counts of the sample assay (EpCAM-expressing targetcells plus PBMCs plus antibody drug) divided by total target cell countsof 5,000 Percentage of cell lysis activity was calculated by multiplyingthe above activity by 100.

In summary, the above Examples demonstrate that the MSFP of the presentdisclosure comprising the 1F3 anti-CD3 Fab, function to redirect T-cellkilling only in the presence of the appropriate tumor target cell (e.g.,EpCAM expressing cells). Surprisingly, anti-EpCAM-OKT3 Fab fusionproteins were not able to bind to Jurkat T cells expressing CD3. Thissuggests that the CD3 epsilon epitope recognized by the 1 F3 Fab isimportant for the function of the 1 F3 Fab fusion proteins. The datadescribed in the above examples supports the use of the MSFPs of thepresent disclosure in a variety of therapeutic settings including in thetreatment of a number of cancers via the recruitment of T cells.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, application and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A bispecific Fab fusion protein comprising: (i) a Fab fragment that binds to the N-terminus of CD3 epsilon (“anti-CD3 Fab fragment”), wherein the anti-CD3 Fab fragment comprises a first chain comprising an immunoglobulin light chain variable (VL) domain and a second chain comprising an immunoglobulin heavy chain variable (VH) domain; and (ii) a fusion moiety A linked to the N-terminus of the VL domain of the anti-CD3 Fab fragment via an optional first linker disposed between the C-terminus of the fusion moiety A and the N-terminus of the VL domain, or a fusion moiety B linked to the N-terminus of the VH domain of the anti-CD3 Fab fragment via an optional second linker disposed between the C-terminus of the fusion moiety B and the N-terminus of the VH domain; wherein the fusion moiety A comprises a cell surface antigen binding domain comprising an antigen binding fragment, and the fusion moiety B comprises a cell surface antigen binding domain comprising an antigen binding fragment; and wherein the VL domain of the anti-CD3 Fab fragment comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; and the VH domain of the anti-CD3 Fab fragment comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO:
 25. 2. The bispecific Fab fusion protein of claim 1, wherein the anti-CD3 Fab fragment binds to an epitope within amino acids 1-27 of CD3 epsilon. 3-9. (canceled)
 10. The bispecific Fab fusion protein of claim 1, wherein each of the antigen binding fragment of the fusion moiety A and the fusion moiety B is an scFv.
 11. (canceled)
 12. The bispecific Fab fusion protein of claim 1, wherein the fusion moiety A and the fusion moiety B bind to a cell surface antigen selected from the group consisting of: FcγRIIb, CD28, CTLA-4, FAS, FGFR1, FGFR2, FGFR3, FGFR4, GITR, LTβR, TLR, TRAIL receptor 1, TRAIL receptor 2, CEA, PSMA, BCMA, CAIX, cMet, EGFR1, Her2/neu, ErbB3, EpCAM, Folate receptor, Ephrin receptor, CD19, CD20, CD30, CD33, CD40, CD37, CD38, and CD138.
 13. (canceled)
 14. The bispecific Fab fusion protein of claim 1, wherein the fusion moiety A and the fusion moiety B are generated from phage display, yeast display, or a human antibody gene transgenic mouse.
 15. (canceled)
 16. The bispecific Fab fusion protein of claim 1, wherein the constant region of the light chain (CL region) of the anti-CD3 Fab fragment comprises a knob or hole mutation, and the heavy chain constant region 1 (CH1 region) of the anti-CD3 Fab fragment comprises a corresponding knob or hole mutation such that the CL region and the CH1 region stably interact.
 17. (canceled)
 18. Isolated polynucleotide or polynucleotides encoding the bispecific Fab fusion protein of claim
 1. 19. An isolated expression vector comprising the isolated polynucleotide or polynucleotides of claim
 18. 20. An isolated host cell comprising the vector of claim
 19. 21. A method of expressing a bispecific Fab fusion protein by culturing the host cell of claim 20 under conditions in which the vector expresses the encoded bispecific Fab fusion protein.
 22. A pharmaceutical composition comprising the bispecific Fab fusion protein of claim 1 and a pharmaceutically acceptable carrier.
 23. A method for treating a cancer in a subject, comprising administering an effective amount of the pharmaceutical composition of claim 22 to the subject having the cancer, wherein the cancer expresses a cell surface antigen to which the bispecific Fab fusion protein can bind.
 24. The bispecific Fab fusion protein of claim 1, wherein the VH domain of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 38, 42, 46, 50, and 54; and/or wherein the VL domain of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 58, 62, 66, and
 70. 25. The bispecific Fab fusion protein of claim 1, wherein the moiety A and the fusion moiety B bind to EpCAM.
 26. The bispecific Fab fusion protein of claim 1, wherein the moiety A and the fusion moiety B bind to CD19.
 27. The bispecific Fab fusion protein of claim 25, wherein each antigen binding fragment of the fusion moiety A and the fusion moiety B: 1) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 139, a CDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 141; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 142, a CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 144; 2) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 147; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 148, a CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 150; 3) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 78 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 78; 4) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 88 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 88; 5) comprises a VH domain comprising amino acid residues 1-116 of SEQ ID NO: 94 and a VL domain comprising amino acid residues 132-241 of SEQ ID NO: 94; or 6) comprises an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 88, and
 94. 28. A bispecific Fab fusion protein comprising: (i) a Fab fragment that binds to the N-terminus of CD3 epsilon (“anti-CD3 Fab fragment”), wherein the anti-CD3 Fab fragment comprises a first chain comprising an immunoglobulin VL domain and a second chain comprising an immunoglobulin VH domain; and (ii) a fusion moiety A linked to the VL domain of the anti-CD3 Fab fragment via an optional first linker disposed between the C-terminus of the fusion moiety A and the N-terminus of the VL domain, or a fusion moiety B linked to the VH domain of the anti-CD3 Fab fragment via an optional second linker disposed between the C-terminus of the fusion moiety B and the N-terminus of the VH domain; wherein the fusion moiety A comprises an EpCAM-binding fragment, wherein the fusion moiety B comprises an EpCAM-binding fragment, and wherein each EpCAM-binding fragment of the fusion moiety A and the fusion moiety B: 1) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 139, a CDR2 comprising the amino acid sequence of SEQ ID NO: 140, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 141; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 142, a CDR2 comprising the amino acid sequence of SEQ ID NO: 143, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 144; 2) comprises a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 147; and a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 148, a CDR2 comprising the amino acid sequence of SEQ ID NO: 149, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 150; 3) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 78 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 78; 4) comprises a VH domain comprising amino acid residues 1-118 of SEQ ID NO: 88 and a VL domain comprising amino acid residues 134-246 of SEQ ID NO: 88; 5) comprises a VH domain comprising amino acid residues 1-116 of SEQ ID NO: 94 and a VL domain comprising amino acid residues 132-241 of SEQ ID NO: 94; or 6) comprises an scFv comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 88, and
 94. 29. The bispecific Fab fusion protein of claim 28, wherein each EpCAM-binding fragment of the fusion moiety A and the fusion moiety B is an scFv.
 30. The bispecific Fab fusion protein of claim 28, wherein the anti-CD3 Fab fragment is humanized.
 31. A pharmaceutical composition comprising the bispecific Fab fusion protein of claim 28 and a pharmaceutically acceptable carrier. 